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The Effects of Different Drying Methods on the In Vitro

Bioaccessibility of Phenolics, Antioxidant Capacity, and Morphology
of European Plums (Prunes domestica L.)
Elif Yener, Oznur Saroglu, Osman Sagdic, and Ayse Karadag*
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ABSTRACT: Four different drying methods, hot-air-drying (HAD), vacuum-drying (VD), ultrasound-assisted vacuum-drying (US-
VD), and freeze-drying (FD), were used to obtain dried plums (Prunes domestica L.). These prunes were evaluated for their physical
properties (such as color, rehydration ratio, and microstructural properties), phenolic compounds, and antioxidant activities before
and after being subjected to in vitro digestion. TPC (total phenolic content) of plums ranged from 196.84 to 919.58 mg of GAE
(gallic acid equivalent)/100 g of dw, and neochlorogenic acid was the most abundant phenolic compound. FD prunes had the
highest levels of phenolics, whereas US-VD caused the most significant loss. During in vitro digestion, the phenolics were present at
higher levels at the gastric medium but failed to maintain their stability at the small intestinal stage. Among the samples, FD along
with HAD prunes exhibited a higher bioaccessibility index for most of the phenolic compounds. The ratios of TPC, TFC (total
flavonoid content), and individual phenolics determined in the digested residues to the initial values of the undigested samples
ranged from 0.23 to 31.03%. It could be concluded that the majority of the phenolics were extracted during digestion. Our findings
showed that the different drying methods would alter the microstructure, which would affect the extractability and release of
phenolics in the simulated digestion model.

1. INTRODUCTION There have been many studies reporting changes in the

phytochemical contents of prunes depending on the drying
The European plum (Prunes domestica) contributes 20% of the
method, such as solar drying,9 freeze-drying,9,10 hot-air-
global plum production.1 Although plums are rich in
drying,9−13 vacuum-drying,10,14 ultrasound-assisted osmotic
nutraceuticals with bioactive properties, their high moisture dehydration,15,16 microwave-vacuum-drying,14 and micro-
content (around 80%) makes them very susceptible to wave-freeze-drying.17 For example, a significant loss of
chemical, physical, and microbiological damage, which neochlorogenic acid9 was reported in solar-dried prunes,
restrains their market availability.2 Drying has been one of whereas in hot-air-dried prunes, the chlorogenic acid loss10
the major preservation methods for extending the shelf life of was more significant. The hot-air-drying resulted in the
fresh fruits. Prunes, the dried form of plums, have been greatest significant decrease in the anthocyanin pigment
reported to contain health-promoting phytochemicals such as
phenolic acids and flavonoids, anthocyanins, dietary fibers, Received: October 24, 2023
sorbitol, and minerals, particularly potassium, iron, magnesium, Revised: February 15, 2024
and calcium.2−4 The consumption of prunes has also been Accepted: February 19, 2024
associated with preventing constipation,5 bone preservation,6,7 Published: March 8, 2024
modulation of the immune response, reduced risk of diabetes,
and progression of atherosclerosis.8
© 2024 The Authors. Published by
American Chemical Society https://doi.org/10.1021/acsomega.3c08383
12711 ACS Omega 2024, 9, 12711−12724
ACS Omega http://pubs.acs.org/journal/acsodf
content (by an average of 82%), along with the total
polyphenol content (by an average of 41%) and chlorogenic
acid (an average of 69%), whereas the anthocyanin loss was an
average of 11 and 10% in the vacuum- and freeze-dried prunes,
respectively.10 It is crucial to choose the appropriate drying
technique because it might negatively affect the nutritional
value of the dried product.2
The phenolic substances should be released from food
matrices during gastrointestinal digestion, become potentially
available for absorption, and exert beneficial health effects on
the human body.18 Previously, it was postulated that the
microstructural changes that occurred during drying would
affect the release of phenolics from food matrices during
digestion and therefore their bioaccessibility.19,20 Drying has
been shown to alter the cellular structure of apple,21
beetroot,22 persimmon,23 mushroom,24 and black Isabel
grapes,25 as well as the release of phenolics and antioxidant
activity in gastrointestinal fluids. Over the years, there have
been some studies evaluating the effects of in vitro gastro-
intestinal (GI) digestion on the bioaccessibility of phenolics
and antioxidant activities in plums.26−30 To the best of our
knowledge, there have been no studies evaluating the in vitro
bioaccessibility of phenolics and antioxidant activities of prunes
dried by different methods. Therefore, in this study, hot-air-,
vacuum-, ultrasound-assisted vacuum-, and freeze-dried prunes
and fresh plums were evaluated in terms of their morphology,
phenolic compounds, and antioxidant activities. In addition,
the effects of in vitro gastrointestinal digestion on the
antioxidant activities and phenolic compounds of the fresh
and dried plums were assessed.
2. MATERIALS AND METHODS
2.1. Chemicals and Reagents. ABTS (2,2-azino-bis(3-
ethylbenzothiazoline)-6-sulfonic acid-diammonium salt,
>98%), DPPH (2,2-diphenyl-1-picrylhydrazyl, 95%) radicals,
Folin−Ciocalteu’s phenol reagent, neocuproine, Trolox (97%),
2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), amylase (A1031),
pepsin (P7012), pancreatin (P7545), bile (B3883), and
analytical standards for HPLC were obtained from Sigma-
Aldrich Ltd. (Steinheim, Germany).
2.2. Drying Procedure. European plum (Prunus domestica
L.) samples were obtained from a local producer in August
2022 in Istanbul, Turkey. The fresh samples with bluish-black
shells and juicy yellow flesh with uniform shape, size, color,
and weight without visible surface damage or diseases selected
for the experiments were washed with water, blotted gently,
and stored at 4 °C for up to 12 h.
Plums as a whole were dried with four different methods;
hot-air-drying (HAD) was done at 60 °C with a constant air
velocity of 1.3 m/s (Memmert UF110, Germany). Vacuum-
drying (VD) was performed in a vacuum drier (Daihan
WOV30, South Korea), regulated by a vacuum pump (EVP
2XZ-2C, Zhejiang, China) with a 60 mbar pressure and 2 L/s
speed. For ultrasound-assisted vacuum-drying (US-VD), the
samples were placed into a conical flask connected to the
vacuum pump and sonication was applied at 20 kHz by the
ultrasonic water bath. Prefrozen samples (−80 °C) were dried
by freeze-drying (FD) (Martin Christ, Germany). The drying
time was 72, 20, 42, and 72 h for HAD, VD, US-VD, and FD,
respectively. Each drying experiment was conducted in
triplicate. The dried prunes were stored at 4 °C in sealed
bags. The moisture content of the samples was 80.02 ± 0.20%
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(fresh), 26.15 ± 0.30% (HAD), 27.08 ± 0.12% (VD), 26.85 ±
0.43% (US-VD), and 26.03 ± 0.60% (FD).
2.3. Proximate Analysis of Plum. The proximate analysis
of fresh plums, including ash, protein (977.02), fat (930.09),
carbohydrate, and dietary fiber (991.43), was determined
according to the Association of Official Analytical Chemists
(AOAC) methods.31 The minerals were analyzed according to
the AOAC method 999 × 1031 by atomic absorption
spectrometry for the determination of different minerals.
2.4. Determination of Color and Rehydration Ratio.
L* (whiteness/darkness), a* (redness/greenness), and b*
(yellowness/blueness) values were measured by a chroma-
meter (Konica Minolta CR-400, NJ). Two grams of the dried
samples were immersed in 100 mL of distilled water at 25 and
50 °C for 420 min. At predetermined time intervals, the
samples were taken out, blotted with tissue paper to eliminate
the excess water on the surface, and weighed. The rehydration
ratio (RR%) was calculated as
RR% = 100 × (mass of rehydrated sample mass of
initial sample)/mass of initial sample
2.5. Scanning Electron Microscopy (SEM). The samples
were cut with a sharp razor blade, mounted on aluminum
specimen stubs by using double-sided tape, and coated with a
thin layer of gold film. The images were taken by an SEM
(QUANTA FEG-250, FEI Company, OR) at 1500 and 2000
magnifications.
2.6. Preparation of Extracts. The plums (∼10 g of fresh
or 2.5 g of dried samples) were homogenized with 80% of
aqueous methanol (1:30, w:v) acidified with 0.1% HCl (v:v) at
10 000 rpm for 5 min (T25 Ultra-Turrax, IKA, NC), held on a
magnetic stirrer overnight, and filtered. The residue was re-
extracted twice, the combined extracts were centrifuged
(2480g, 10 min, 4 °C), and the supernatant was evaporated
under vacuum and then reconstituted to 6 mL with the
extraction solvent and stored at −18 °C.
2.7. Total Phenolic, Total Flavonoid, and Total
Anthocyanin Contents. The total phenolic content (TPC)
of the samples was determined with the Folin−Ciocalteu (FC)
reagent method according to the method described by
Singleton and Rossi.32 An aliquot of 0.5 mL of the extracts
was added to 2.5 mL of the FC reagent (0.2N) and 2 mL of a
Na2CO3 (7.5%) solution. The mixture was incubated at room
temperature for 30 min in the dark. The absorbance was
measured at 760 nm using a Shimadzu 150 UV-1800
spectrophotometer (Kyoto, Japan).
The total flavonoid content (TFC) of the samples was
determined according to the method described by Zhishen et
al.33 The extract (1 mL) was mixed with 4 mL of distilled
water, 0.3 mL of NaNO2 (5%), and 0.3 mL of an AlCl3 (10%)
solution, and the mixture was left for 6 min. Then, 2 mL of
NaOH (1 M) was added, and the volume was completed to 10
mL with distilled water. The absorbance was measured at 510
nm by a Shimadzu UV-1800 spectrophotometer.
The total monomeric anthocyanin (TA) content was
determined according to the pH differential method of Giusti
and Wrolstad.34 Absorbance was measured at 510 and 700 nm.
The TA amount was determined by using the following
equation
A = (A510 A 700)pH1.0 (A510 A 700)pH4.5
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TA (mg/L) = (A × MW × DF × 1000)/( × L)
A is the absorbance at 510 and 700 nm, MW: molecular weight
for cyanidin 3-O-glucoside (449.2 g/mol), ε: molar extinction
coefficient for cyanidin 3-O-glucoside (26900), DF: dilution
factor, and L: cell path length (1 cm).
The results of TPC, TFC, and TA were expressed as mg of
gallic acid equivalent (GAE)/100 g of dw, mg of catechin
equivalent (CE)/100 g of dw, and mg of cyanidin 3-O-
glucoside (cy-3-O-glc)/100 g of dw, respectively.
2.8. Antioxidant Activity Assessment. The DPPH
radical scavenging activity assay was carried out according to
the method of Brand-Williams et al.35 Volumes of 4.9 mL of
DPPH solution (0.1 Mm) and 0.1 mL of the extracts were
mixed and incubated for 20 min in the dark at room
temperature, and the absorbance was measured at 517 nm by a
Shimadzu UV-1800 spectrophotometer. The cupric-reducing
antioxidant capacity (CUPRAC) was performed as described
by Apak et al.36 The 1 mL portions of CuCl2 (0.01 M),
neocuproine (7.5 mM), and ammonium acetate buffer (1 M,
pH 7.0) were mixed. After the addition of 0.1 mL of the extract
and 1 mL of distilled water; the mixture was incubated at room
temperature for 1 h in the dark. The absorbance was measured
at 450 nm. ABTS radical scavenging activity was determined
using the method described by Re et al.37 After adding 2 mL of
diluted ABTS solution to a 0.1 mL extract, it was left in the
dark for 6 min and the absorbance was measured at 734 nm.
The FRAP (ferric reducing antioxidant power) assay was
performed according to Benzie and Strain.38 A 100 μL portion
of the extract was mixed with 900 μL of water and 2 mL of the
FRAP reagent and incubated at room temperature for 30 min
in the dark. The absorbance was measured at 593 nm. All
results of antioxidant activity assessments were given as mmol
of Trolox equivalent (TE)/100 g of dw.
2.9. HPLC Analysis of Phenolic Compounds. Phenolic
compounds were determined using the HPLC system (LC-
20AD pump, SIL-20A HT autosampler, CTO-10ASVP column
oven, DGU-20A5R degasser, and CMB-20A communications
bus module) coupled to a diode array detector�SPDM20A
DAD (Shimadzu Corp., Japan) according to Karadag et al.39
Separations were conducted at 40 °C on an Inertrsil ODS C-
18 reversed-phase column (250 mm × 4.6 mm, 5 μm particle
size, GL Sciences, Japan). The individual anthocyanins were
determined according to Capanoglu et al.40 The mobile phases
were acetic acid in water (0.1:99, v:v) and acetic acid in
acetonitrile (0.1:99, v:v). A gradient elution was applied as
follows: 10% B (0−2 min), 10−30% B (2−27 min), 30−90% B
(27−50 min), and 90−100% (51−60 min) and at 63 min
returns to the initial conditions. Chromatograms were acquired
at 278, 320, 360, and 520 nm to quantify phenolic acids,
flavonoids, and anthocyanins. Identification and quantitative
analyses were performed by comparing UV absorption spectra
and retention times of each compound with those of external
standards. The stock solutions of reference standards (gallic
acid, caffeic acid, chlorogenic acid, neochlorogenic acid, ellagic
acid, ferulic acid, rutin, quercetin, cyanidin 3-O-glucosides, and
peonidin 3-O-glucosides) were prepared in methanol. The
working solutions at concentrations ranging from 1 to 100 mg/
L were achieved through the dilution of the stock solution with
methanol. Calibration curves were generated by graphing the
peak areas of the compounds identified relative to the peak
areas against the concentration of the standard solution. The
calibration curves based on duplicate injections demonstrated
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good linearity, with R2 values exceeding 0.99 (peak area vs
concentration). All analyses were performed in triplicate, and
the results were given as mg/100 g dw.
2.10. In Vitro-Simulated Digestion Procedure. In vitro-
simulated digestion assay was performed according to
Brodkorb et al.41 and Minekus et al.42 The fresh and dried
plums were mixed 1:1 (w: (v) with simulated salivary fluid
(SSF), α-amylase (75 U/mL), and CaCl2 (0.75 mM)) and
vortexed for 2 min at 37 °C (pH 7.0). The oral bolus was
diluted with simulated gastric fluid (SGF) (1:1, v:v), CaCl2
(0.075 mM), and pepsin (2000 U/mL) and incubated at 100
rpm for 2 h at 37 °C (pH 3.0). The gastric chyme was mixed
with simulated intestinal fluid (SIF) (1:1, v:v), CaCl2 (0.3
mM), pancreatin (100 U/mL), and fresh bile (10 mM) at pH
7.0. The segments of dialysis bags (MWCO 12 kDa) filled with
NaHCO3 (0.1 M) were placed in SIF medium, and the beakers
were kept at 100 rpm for 2 h at 37 °C.43 The content of the
dialysis bags was the compounds that entered the serum (IN),
and those outside the bags were the material that remained in
the GI tract (OUT). The supernatants taken for the oral,
gastric, and intestinal phases were collected after centrifugation
at 2480g, 10 min, 4 °C, filtered (0.45 μm), immediately frozen
in liquid nitrogen, and lyophilized. The powders of lyophilized
oral, gastric, and intestinal phases were redissolved in the
extraction solvent (80% of aqueous methanol acidified with
0.1% HCl). A blank test tube without samples but with all
digestion fluids was also subjected to analysis. The solid
residue that remained after centrifugation at the end of
intestinal digestion was collected as a nonbioaccessible
fraction, and its methanolic extract was prepared (2.6). All
procedures were performed in triplicate. Bioaccessibility index
(BI%) and recovery percentage (R%) were calculated as25,44
Cintestinal (IN + OUT)
BI % = × 100
Cnondigested (initial)
C IN
R%= × 100
Cnondigested (initial)
2.11. Statistical Analysis. All experiments were carried
out in triplicate, and the data were reported as the mean ± the
standard deviation. Statistical analysis was performed with
SPSS Statistics Software (IBM version 20). One-way ANOVA
Tukey’s post hoc test was applied to the responses (measured
bioactive properties) to determine the differences among
samples (the prunes obtained by four different drying methods
and fresh plum).The differences in responses observed for each
sample that was subjected to in vitro gastrointestinal digestion
were compared among digestion phases. The calculated values
of R% and BI% at the end of the simulated digestion phase
were compared among the samples.25 Differences were
considered significant if p < 0.05. A heat map was constructed
with Origin Pro 2023 statistical analysis software (Origin Lab
Corp., MA) to better illustrate the data (the change of
individual phenolic compounds, TPC, TFA, TA, and
antioxidant activities at each digestion stage).45
3. RESULTS AND DISCUSSION
3.1. Proximate Composition of Fresh Plum. The dry
matter content of fresh plum was 19.98 ± 0.2%, and the
carbohydrate and dietary fiber contents were 16.90 ± 0.21 and
1.86 ± 0.05 g per 100 g of fresh plum, respectively. Plums were
considered high-fiber fruits; the dietary fiber content was
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Table 1. Total Phenolic Content (TPC), Total Flavonoid Content (TFC), Total Anthocyanin (TA), and Antioxidant Activities
and Individual Phenolics of Fresh and Dried Plumsa
fresh HAD VD US-VD FD
b b c c d
TPC 656.91 ± 17.49 498.23 ± 23.11 504.41 ± 58.78 196.84 ± 7.27 919.58 ± 28.81a
TFCc 247.10 ± 19.53b 276.53 ± 18.75b 172.00 ± 2.20c 39.56 ± 2.03d 432.34 ± 36.54a
TAd 50.28 ± 4.19b 1.71 ± 0.01c 59.47 ± 9.36b 0.72 ± 0.20c 87.01 ± 5.21a
e
Antioxidant Activity (mmol TE/100 g dw)
DPPH 2.16 ± 0.10b 1.26 ± 0.03c 2.33 ± 0.19b 0.48 ± 0.01d 3.09 ± 0.05a
CUPRAC 5.39 ± 0.62b 3.58 ± 0.16c 4.34 ± 0.15c 1.06 ± 0.14d 7.66 ± 0.37a
ABTS 5.41 ± 0.54b 2.34 ± 0.27 c
3.55 ± 0.07c 0.55 ± 0.05d 6.45 ± 0.08a
FRAP 2.84 ± 0.34b 1.93 ± 0.10 c
2.94 ± 0.15b 0.44 ± 0.01d 4.06 ± 0.19a
Phenolics (mg/100 g dw)
gallic acid 11.05 ± 1.15a 6.18 ± 0.76b 6.74 ± 0.01b 1.74 ± 0.01c 6.76 ± 0.17b
neochlorogenic acid 93.74 ± 6.47b 54.30 ± 1.85 c
62.59 ± 2.58c 1.47 ± 0.10d 155.09 ± 0.23a
chlorogenic acid 51.13 ± 8.09b 32.55 ± 5.20 c
24.29 ± 1.51c 0.98 ± 0.06d 80.34 ± 4.58a
caffeic acid 7.08 ± 0.29a 4.69 ± 0.01b 3.65 ± 0.00c 0.95 ± 0.20d 4.60 ± 0.23b
ellagic acid 13.38 ± 1.26a 6.94 ± 0.02b 7.65 ± 0.13b 1.56 ± 0.01c 7.63 ± 0.19b
ferulic acid 1.62 ± 0.01a 1.53 ± 0.02 b
0.85 ± 0.00d 0.39 ± 0.00e 0.89 ± 0.00c
quercetin 28.99 ± 0.02a 15.87 ± 0.00 c
15.93 ± 0.00b nd 15.85 ± 0.00c
rutin 7.25 ± 0.74b 3.42 ± 0.07c 5.75 ± 0.24c 0.47 ± 0.02d 12.01 ± 1.16a
Anthocyanins (mg/100 g dw)
Cyn-3-O-glc 34.45 ± 5.39b nd 24.45 ± 0.28c nd 45.14 ± 3.27a
Pn-3̅-O-glc 12.83 ± 1.61a nd 8.17 ± 0.15b nd 13.82 ± 1.94a
a
Data are expressed as mean ± SD of triplicate measurements. Means with different letters in the same row are significantly different (p < 0.05).
HAD, hot-air-drying; VD, vacuum-drying; US-VD, ultrasound-assisted vacuum-drying; FD, freeze-drying. bTPC, total phenolic content (mg GAE/
100 g dw). cTFC, total flavonoid content (mg CE/100 g dw). dTA; total anthocyanin content (mg cyanidin 3-O-glucoside (cyn-3-O-glc)/100 g
dw). eDPPH, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity; CUPRAC, copper reducing antioxidant capacity; ABTS, 2,2′-azino-bis (3-
ethylbenzothiazoline)-6-sulfonic acid radical scavenging activity and FRAP, ferric reducing antioxidant power, TE, Trolox equivalent; Cyn-3-O-glc,
cyanidin 3-O-glucoside; Pn-3̅-O-glc, peonidin 3-O-glucoside; nd, not detected.

around 1.5 g per 100 g of fresh plums and can be elevated to
6.5 g per 100 g of dried fruits.4 Potassium (227.34 ± 2.96 mg/
100 g) was the major mineral in our sample, followed by
phosphorus, magnesium, and calcium (Table S1), and those
levels were comparable to the previous studies.8
3.2. Physical Properties of Fresh and Dried Plums. In
addition to being a quality indicator for consumer acceptance,
the color of dried fruits was also associated with the content of
anthocyanins and other phenolic compounds. The whiteness
of prunes (L*) was increased by FD on the outer and inner
surfaces; the other drying methods did not cause any
considerable change in L* values on the outer surface but
yielded darker colors on the inner surfaces (Table S2).
The increase in the lightness of freeze-dried fruits has been
reported previously as related to the porous structure and
existence of air voids and the difference in light diffusion that
passes through the sample due to the replacement of water
with air.46,47 The surface of fresh plum had purple tints with
positive a* (redness) and negative b* (blueness) values, and
those were generally reduced by drying, except for FD samples.
US-VD yielded prunes with a more dull color and had the
lowest a* and b* values. The color of the plum surface might
also be related to the presence of anthocyanins, and the highest
anthocyanin loss was determined in US-VD samples, followed
by HAD (Table 1). The positive b* value (yellowness)
observed on the inner surface of the fresh plum was
significantly reduced by HAD and US-VD. On the contrary,
compared to the fresh sample, similar to the lightness value,
FD prunes presented a higher yellowness. The inner surface a*
value of fresh plum was negative, indicating more green tints,
and by drying, all samples presented positive a* values with
hues of redness. The highest redness was observed in HAD
12714
and VD prunes, whereas in FD and US-VD samples, the a*
value was similar. The increase in redness on the interior
surface might be related to enzymatic and nonenzymatic
reactions influenced by the presence of oxygen and the
duration of heating. It has been reported that during hot-air-
drying of plum varieties, the sucrose was hydrolyzed to
reducing sugars that take part in Maillard browning reactions
through the action of invertase and organic acids released due
to the disruption of the cell membrane.48
The rehydration ratio is related to the extent of structural
drying, and generally, a higher rehydration rate means less
shrinkage and well-defined voids.49,50 FD samples showed the
highest rehydration at both temperatures (25 and 50 °C), and
the lowest ratio was observed in HAD and US-VD prunes
(Figure S1). It is known that freeze-dried fruits have higher
porosity and the least shrinkage, which would enhance water
absorption.47 The lower rehydration of US-VD and HAD
samples may indicate the presence of more shrunken and
damaged cells and capillaries that lowered the intake of water.
Additionally, the thick layer formed on the outer surface of
plums by US-VD and HAD may also jeopardize the
rehydration ratio.
3.3. Microstructural Changes. The surface of fresh plums
(Figure 1A1) was smooth, and the presence of stomata (white
arrows) that allow the passage of gas and the microwrinkles
caused by the wax layer (black arrows) could be noticed. The
cross section and the inner surface of the fresh plum (Figure
1A2) had larger parenchymal cells, showing no shrinkage and
the presence of voids due to the high water content. Among
the outer surfaces of dried plums, the ones obtained by FD
(Figure 1B1) and VD (Figure 1C1) most resemble the surface
of fresh plums, and the clustering of the wax layer due to
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Figure 1. Morphology of fresh plum (A), prunes dried by FD (B), VD (C), HAD (D), and US-VD (E). FD, freeze-drying; VD, vacuum-drying;
HAD, hot-air-drying; and US-VD, ultrasound-assisted vacuum-drying. 1 and 2 correspond to the outer surface (2000x magnification) and the cross
section (1500× magnification) of the samples, respectively.

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dehydration and the presence of some cracks can be seen. The
HAD plums (Figure 1D1) showed fractured surfaces with
separate flaked layers. The drying of plums at high temper-
atures may cause the degradation of polysaccharides,
alterations in the bonding between polymers, and the
separation of the cells. In US-VD prunes (Figure 1E1), the
surface had high levels of microgranular aggregates. In HAD
and US-VD, the application of heat and ultrasound caused
more microstructural stress due to the longer drying time and
the application of ultrasonic cavitation. The cross-sectional
images of dried samples presented more porous structures;
however, their size, depth, and distribution varied by the
methods of drying. In FD and VD (Figure 1B2,C2), larger
hollow openings can be observed, whereas for HAD and
especially US-VD samples, the voids and holes became shallow
and distorted (Figure 1D2,E2). These results were also
associated with the lower rehydration ratio of dried plums
obtained by HAD and US-VD.
3.4. Total Phenol, Total Flavonoid Content, Anti-
oxidant Activity, and Phenolic Composition of Fresh
and Dried Plums. The total phenolic content (TPC) and
total flavonoid content (TFC) of fresh plum were 656.91 ±
17.49 mg of GAE/100 g of dw and 247.10 ± 19.53 mg of CE/
100 g of dw, respectively. Our results were in agreement with
the results of Miletić et al.51 who determined the TPC of three
blue-purple plum varieties between 215 and 643 mg GAE/100
g fw, and the TFC value ranged from 100 to 345 mg CE/100 g
dw. Kaulmann et al.52 determined the TPC level in the range
of 111−170 mg GAE/100 g and 27−76.5 mg CE/100 g dw in
some European plum varieties (President, Italian, and Kirkes
blue plums). The total anthocyanin content (TA) of our
sample was 50.28 ± 4.19 mg cyn-3-O-glc/100 g dw, and it was
determined between 5.59 and 12.8 mg cyn-3-O-glc/100 g fw in
the similar plum varieties.52
Gościnna et al.10 studied the bioactive properties of three
different blue-purple skin plums (Bluefree, Stanley, and Sweet
Common) and when compared to our results, they found
lower TPC values (173−225 mg GAE/g dw) but higher TA
content (82−148.3 mg cyn-3-O-glc/100 g dw) in their three
plum cultivars.
All drying methods, except for FD, caused a reduction in
TPC levels compared with fresh samples. The highest
reduction was observed under US-VD conditions, while the
difference between HAD and VD was not significant. Similarly,
US-VD-treated plums had the lowest TFC value, while HAD
prunes had a higher amount of TFC, comparable to that of the
fresh sample. The TA value of VD prunes was similar to that of
fresh samples, whereas HAD and US-VD samples showed a
dramatic decrease (around 98%). Our results were consistent
with Gościnna et al.,10 who determined that HAD at 60 °C
resulted in the greatest significant decrease in TA (by an
average of 82%) and TPC (by an average of 41%). Reductions
or losses of phenolic compounds by hot-air-drying have been
associated with thermal and enzymatic degradation, especially
when oxygen is present.53
The drying temperature in our study was 60 °C, and the
drying time was in HAD (72 h), followed by that in US-VD
(42 h) and VD (20 h). Although the drying time of HAD was
higher than that of US-VD, the highest reduction of TPC,
TFC, and TA was observed in US-VD. Due to the alternating
compressions and expansions of the material during sonication,
the water inside the material could move the surface through
the microscopic channels; however, the crystalline waxy outer
12716
Article
surface of the plum may not provide enough rubbery state to
allow this water transfer to the surrounding environment and
therefore could restrict the mobility of water.54 It may cause
the creation of localized hotspot regions in the fruit that also
trigger the loss of active compounds. The use of freeze-drying
has been shown to significantly enhance the extractability of
phenolics in blueberries,55 goji berries,56 and stinging nettle.57
The increase in the measured bioactive properties in FD
prunes compared to that in fresh samples could be related to
the enhanced extractability of those compounds into the
extraction solvent due to the mild microstructural changes
induced by the ice-crystal formation during freeze-drying and
higher rehydration with the solvent due to the porous
structure.
As shown in Table 1, the antioxidant activity of the samples
followed a trend similar to the TPC, TFC, and TA values. The
highest antioxidant activity values were observed in FD prunes,
which were generally followed by fresh samples, and US-VD
prunes displayed the lowest values. The difference between
fresh and VD samples was not significantly different while
evaluating antioxidant activity by FRAP and DPPH assays. The
sum of all individual phenolics quantified by HPLC-DAD
analysis was lower than the TPC value attained by the Folin−
Ciocalteu spectrophotometric assay. Despite its popularity, the
Folin−Ciocalteu test is not specifically designed for phenolic
compounds, as the reagent could also be reduced by other
nonphenolic compounds such as ascorbic acid, sugar, amino
acid, and redox-active metal ions present in the sample, with
the risk of content overestimation.58 HPLC analysis is more
sensitive and specific to target phenolic compounds, and the
quantification of individual phenolics was based on a
comparison of the eluted peaks with the available reference
standards.
Although they were not determined in our study, plums
were reported to contain proanthocyanidin derivatives, such as
the dimers procyanidin B1, B4, and B2. When five different
plum cultivars were analyzed at different stages of maturation,
although catechin or epicatechin was not detected in
significant amounts, dimeric and trimeric forms of catechin
were quantified, particularly in the skins of the fruit.59
In fresh plum, among phenolic acids, neochlorogenic acid
(93.74 ± 6.47 mg/100 g dw) was the major compound,
followed by chlorogenic acid (51.13 ± 8.09 mg/100 g dw),
ellagic, caffeic, and gallic acids. Our results were consistent
with Piga et al.13 as the neochlorogenic and chlorogenic acid
contents in the Stanley cultivar were 381.77 and 54.51 mg/100
g dw, higher than the values determined by Gościnna et al.10
(111.7 mg and 42.5 mg/100 g dw, respectively). Kim et al.60
reported the contents of neochlorogenic acid (from 18.1 to
215.4 mg/100 g fw) and chlorogenic acid (0.9−21.0 mg/100 g
fw) in the fresh fruits of 11 plum cultivars. The amount of
chlorogenic and neochlorogenic acid contents was significantly
reduced by drying, except for FD samples. The highest
reduction (more than 95%) was observed in the US-VD
prunes. HAD and VD drying resulted in a 33−42% reduction
in neochlorogenic acid and up to a 50% reduction in the
chlorogenic acid content. The amounts of those compounds
determined in HAD and VD drying were not significantly
different from each other. The decrease in the chlorogenic acid
content could be due to the formation of neochlorogenic and
cryptochlorogenic acid isomers, which occurred through
isomerization due to molecular lactone migration at higher
temperatures,61 whereas in FD prunes, a higher amount of
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Table 2. Change in the Total Phenolic Content (TPC), Total Flavonoid Content (TFC), Total Anthocyanin (TA), and
Antioxidant Activities of Fresh and Dried Plums during In Vitro Digestiona,b
intestinal
oral gastric IN OUT R% BI %
TPC
fresh 88.04 ± 7.51c 349.03 ± 45.78a 17.93 ± 2.49d 193.11 ± 22.03b 2.72 ± 0.32B 32.19 ± 3.93C
HAD 103.18 ± 7.78b 230.29 ± 18.84a 22.65 ± 2.59c 235.64 ± 1.71a 4.53 ± 0.36A 51.89 ± 1.85B
VD 86.36 ± 5.98b 132.76 ± 14.10a 2.37 ± 0.40c 62.60 ± 5.89b 0.48 ± 0.13C 13.23 ± 4.49D
US-VD 24.78 ± 2.74c 142.75 ± 6.92b 5.30 ± 1.54c 217.31 ± 17.97a 2.71 ± 0.84B 113.01 ± 4.90A
FD 139.80 ± 15.13c 355.35 ± 26.57a 17.35 ± 1.44d 253.27 ± 65.34b 1.89 ± 0.21B 29.50 ± 7.66C
TFC
fresh 22.01 ± 2.10b 281.18 ± 3.02a 3.49 ± 1.04c 5.62 ± 2.47c 1.41 ± 0.38B 3.64 ± 1.09B
HAD 29.77 ± 1.32b 68.07 ± 0.33a 2.39 ± 0.38d 11.50 ± 3.22c 0.86 ± 0.08BC 4.99 ± 1.00B
VD 20.66 ± 1.02b 37.38 ± 3.01a 1.35 ± 0.69d 12.93 ± 0.81c 0.78 ± 0.39BC 8.30 ± 0.39B
US-VD 7.52 ± 1.48b 33.80 ± 2.93a 1.11 ± 0.10c 5.13 ± 1.33bc 2.83 ± 0.31A 15.95 ± 4.23A
FD 59.82 ± 1.01c 243.46 ± 7.27a 2.67 ± 0.17d 88.72 ± 2.65b 0.61 ± 0.01C 21.20 ± 1.24A
TA
fresh 5.76 ± 2.38b 16.40 ± 3.62a 0.33 ± 0.08b 1.73 ± 0.57b 0.67 ± 0.21B 4.19 ± 1.59B
HAD 0.09 ± 0.02b 0.13 ± 0.04b 0.42 ± 0.13a 8.17 ± 2.72B 33.30 ± 7.08B
VD 1.69 ± 0.62ab 2.55 ± 0.38a 0.01 ± 0.00c 1.49 ± 0.08b 0.02 ± 0.00B 2.59 ± 0.56B
US-VD 1.06 ± 0.16a 0.10 ± 0.04c 0.12 ± 0.01c 0.61 ± 0.10b 18.49 ± 6.42A 109.15 ± 38.41A
FD 2.08 ± 1.17b 28.95 ± 8.46a 0.13 ± 0.02b 2.45 ± 0.44b 0.11 ± 0.09B 2.98 ± 0.57B
DPPH
fresh 370.16 ± 46.25a 207.66 ± 15.06b 115.60 ± 35.25c 51.83 ± 12.58c 5.32 ± 1.47A 7.74 ± 1.34C
HAD 130.73 ± 34.07b 185.89 ± 6.53a 50.71 ± 2.28c 88.63 ± 6.74bc 4.02 ± 0.07AB 11.05 ± 0.42B
VD 171.96 ± 49.83a 73.79 ± 9.90b 14.68 ± 1.94b 59.40 ± 19.37b 0.63 ± 0.07C 3.22 ± 1.01D
US-VD 108.52 ± 10.96a 16.81 ± 8.56c 9.86 ± 3.13c 67.42 ± 9.70b 2.03 ± 0.59BC 16.01 ± 1.68A
FD 250.01 ± 19.10b 1830.10 ± 34.24a 121.39 ± 17.75c 72.46 ± 12.10c 3.92 ± 0.57AB 6.27 ± 0.52C
CUPRAF
fresh 668.58 ± 33.57c 1750.25 ± 108.02a 177.65 ± 24.10d 856.91 ± 51.07b 3.30 ± 0.44B 19.35 ± 2.81C
HAD 558.56 ± 56.06b 1387.32 ± 53.82a 93.32 ± 15.22c 677.28 ± 100.33b 2.61 ± 0.50B 21.48 ± 1.46C
VD 590.52 ± 58.95b 617.87 ± 2.46b 30.17 ± 6.38c 831.39 ± 111.35a 0.69 ± 0.14C 19.80 ± 2.09C
US-VD 243.26 ± 48.76b 681.62 ± 86.78a 66.50 ± 3.81c 585.73 ± 48.28a 6.30 ± 0.58A 61.94 ± 8.09A
FD 901.02 ± 46.07b 3592.00 ± 247.93a 177.30 ± 15.77c 3156.6 ± 416.92a 2.31 ± 0.10B 43.48 ± 4.34B
ABTS
fresh 378.14 ± 95.78bc 3856.29 ± 241.48a 142.66 ± 20.00c 569.70 ± 78.04b 2.65 ± 0.47BC 13.21 ± 2.50BC
HAD 195.28 ± 58.99b 530.79 ± 12.20a 44.91 ± 24.32c 76.30 ± 10.69c 1.93 ± 0.37BC 5.21 ± 0.56C
VD 200.00 ± 19.01b 396.85 ± 16.69a 107.03 ± 20.53c 453.77 ± 37.57a 3.01 ± 0.63B 15.77 ± 1.23BC
US-VD 59.05 ± 8.91c 368.84 ± 54.58a 72.38 ± 7.36c 204.75 ± 37.99b 12.96 ± 0.77A 50.95 ± 9.74A
FD 344.59 ± 196.5b 1549.67 ± 15.01a 94.49 ± 19.53b 1345.91 ± 298.9a 1.46 ± 0.32C 22.30 ± 4.61B
FRAP
fresh 261.97 ± 0.96b 3011.38 ± 100.46a 55.01 ± 4.64c 215.02 ± 2.54b 1.97 ± 0.37C 9.62 ± 1.21C
HAD 198.66 ± 32.61b 430.78 ± 36.14a 107.10 ± 1.04b 438.43 ± 101.13a 5.55 ± 0.34A 28.46 ± 6.76B
VD 127.25 ± 3.98b 414.80 ± 15.20a 4.11 ± 0.20d 94.07 ± 9.57c 0.14 ± 0.01D 3.34 ± 0.22C
US-VD 120.89 ± 8.09b 466.19 ± 27.72a 11.80 ± 1.04c 90.82 ± 14.83b 2.70 ± 0.26B 23.53 ± 3.11B
FD 514.49 ± 26.33c 2734.13 ± 43.09a 113.26 ± 5.57d 1590.71 ± 130.5b 2.78 ± 0.08B 42.08 ± 4.55A
a
Data are expressed as mean ± SD of triplicate measurements. Different lowercase letters in the same row for each sample are significantly different
(p < 0.05) among digestion phases. Different capital letters on the same column for R% and BI% are significantly different (p < 0.05) among prune
samples. R% (recovery percent) = CIN/Cnondigested × 100 BI%, (bioaccessibility index) = Cintestinal (IN + OUT)/Cnondigested. bHAD, hot-air-drying;
VD, vacuum-drying; US-VD, ultrasound-assisted vacuum-drying; FD, freeze-drying; TPC, total phenolic content (mg GAE/100 g dw); TFC, total
flavonoid content (mg CE/100 g dw); TA, total monomeric anthocyanins (mg cyn-3-O-glc/100 g dw); DPPH, 2,2-diphenyl-1-picrylhydrazyl
radical scavenging activity; CUPRAC, copper reducing antioxidant capacity; ABTS, 2,2′-azino-bis (3-ethylbenzothiazoline)-6-sulfonic acid radical
scavenging activity; and FRAP, ferric reducing antioxidant power (μmol TE (Trolox equivalent)/100 g dw).

chlorogenic acid derivatives was detected compared to the
fresh sample. During FD, bioactive compounds are expelled
from the epithelial cells at a higher rate, and the release of
polyphenol compounds might be increased due to the
breakdown of cellular constituents.62 Among flavonoids, rutin
(quercetin 3-rutinoside) was determined in our samples.13,51,60
The rutin content of fresh plums was significantly higher than
that of dried samples except FD prunes. The presence of
12717
anthocyanins such as cyanidin 3-O-glucoside, cyanidin 3-O-
rutinoside, peonidin 3-O-glucoside, and peonidin 3-O-rutino-
side10,60,63 in plums has been previously reported. By
comparison with the available external standards we had,
cyanidin 3-O-glucoside (34.45 ± 5.39 mg/100 g of dw) and
peonidin 3-O-glucoside (12.83 ± 1.61 mg/100 g of dw) were
detected and quantified in our sample (Table 1). Only FD and
VD samples had anthocyanin constituents, and compared to
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Table 3. Total Phenolic Content (TPC), Total Flavonoid Content (TFC), Total Anthocyanin (TA), and Antioxidant Activities
and Individual Phenolics of Nonbioaccessible Fraction�the Digested Residue Remained after In Vitro Digestiona
fresh HAD VD US-VD FD
b b a a b b
TPC 22.82 ± 2.27 72.23 ± 9.35 72.19 ± 8.69 14.52 ± 0.19 10.92 ± 1.16
3.47% 14.50% 14.30% 7.37% 1.18%
TFCc 0.93 ± 0.53c 3.39 ± 0.47b 4.57 ± 0.40ab 0.96 ± 0.24c 5.43 ± 0.80a
0.37% 1.22% 2.68% 2.42% 1.25%
TAe 0.96 ± 0.28a 0.85 ± 0.12a
1.94% 0.97%
Antioxidant Activityd (μmol TE/100 g dw)
c
DPPH 14.60 ± 7.39 57.28 ± 22.25ab 20.16 ± 1.25c 27.21 ± 7.57bc 69.61 ± 13.36a
0.67% 4.54% 0.86% 5.66% 2.25%
CUPRAC 97.98 ± 15.70b 153.20 ± 4.76a 147.98 ± 13.15a 163.99 ± 22.86a 152.91 ± 1.88a
1.81% 4.27% 3.40% 15.47% 1.99%
ABTS 53.37 ± 5.82a 13.83 ± 1.41b 11.76 ± 1.31b 21.53 ± 6.09b 12.06 ± 0.38b
0.98% 0.59% 0.33% 3.98% 0.18%
FRAP 30.68 ± 0.76c 194.10 ± 5.47b 49.77 ± 0.28c 37.14 ± 1.49c 241.14 ± 15.87a
1.08% 10.05% 1.69% 8.44% 5.93%
Phenolics (mg/100 g dw)
gallic acid 1.06 ± 0.01a 0.39 ± 0.01c 0.40 ± 0.01c 0.54 ± 0.01b 0.31 ± 0.02d
9.59% 6.31% 5.93% 31.03% 4.58%
neochlorogenic acid
chlorogenic acid 0.12 ± 0.00c 0.46 ± 0.13a 0.25 ± 0.05ab 0.23 ± 0.00ab 0.35 ± 0.14ab
0.23% 1.43% 1.02% 23.71% 0.43%
caffeic acid 0.17 ± 0.00b 0.14 ± 0.00c 0.20 ± 0.01a 0.18 ± 0.02b
3.62% 3.83% 21.27% 3.91%
ellagic acid 0.24 ± 0.00b 0.28 ± 0.00b 0.38 ± 0.04a
3.45% 3.66% 4.98%
ferulic acid 0.11 ± 0.00a 0.06 ± 0.00b 0.11 ± 0.00a 0.04 ± 0.00b
7.18% 7.05% 28.20% 4.49%
quercetin 0.63 ± 0.03b 0.65 ± 0.02b 0.63 ± 0.05b
3.96% 4.08% 3.97%
rutin 0.05 ± 0.03c 0.19 ± 0.06b 0.07 ± 0.00c 1.02 ± 0.02a
1.46% 3.86% 14.89% 8.49%
a
Data are expressed as mean ± SD of triplicate measurements. The percentage values (%) indicate the ratio of the measured value remained in the
digested residue to the value of the undigested sample. Means with different letters in the same row are significantly different (p < 0.05). HAD, hot-
air-drying; VD, vacuum-drying; US-VD, ultrasound-assisted vacuum-drying; and FD, freeze-drying. bTPC, total phenolic content (mg GAE/100 g
dw). cTFC, total flavonoid contents (mg CE/100 g dw). dDPPH, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity; CUPRAC, copper
reducing antioxidant capacity; ABTS, 2,2′-azino-bis (3-ethylbenzothiazoline)-6-sulfonic acid radical scavenging activity, FRAP, ferric reducing
antioxidant power, TE, Trolox equivalent. eTA, total monomeric anthocyanins (mg cyn-3-O-glc/100 g dw).

fresh samples, their level was reduced in VD prunes (29−36%
reduction).
3.5. Effect of Drying Methods on the In Vitro
Bioaccessibility of Plum Phenolics and Antioxidant
Activity. The change in TPC, TFC, TA, and antioxidant
activity values at various stages of in vitro gastrointestinal
digestion, including the oral, gastric, and intestinal phases, was
given in Table 2. TPC, TFC, and TA levels in the oral phase
were lower compared with the initial levels found in the
methanolic extract (Table 1) of undigested samples. This
decrease can be attributed to the limited solubility of these
compounds in simulated saliva fluid and the relatively short
duration of this step (2 min). Among the dried plums, FD
prunes exhibited the highest release of TPC and TFC,
potentially due to their porous structure, which could enhance
extractability in the oral bolus. Generally, in all samples, TPC,
TFC, CUPRAC, ABTS, and FRAP values increased at the
gastric phase. In previous studies, an increase in the amount of
biologically accessible polyphenols and flavonoids has been
observed during gastric digestion.28,64−66 The release of
polyphenols from apples after simulated gastrointestinal
12718
digestion was mainly achieved in the gastric phase. However,
their content was still significantly lower than that obtained by
the methanol extraction.67 Although it was reported that most
of the phenolic compounds in plum fruits were present in free
form, the ratio of free phenolics to the total phenolic content
(free+bound) was reported to be around 70% in Natal plum,30
92%,29 and 80−90%27 in Japanese plum varieties. When Seke
et al.30 studied the effect of free and bound phenolic
compounds on the antioxidant activity of plum fruit, they
concluded that although most of the phenolic compounds
were present in free form (939.03 mg GAE/kg fw), TPC in the
bound insoluble fraction of plum fruits was higher after acid
hydrolysis (255.5 mg GAE/kg fw) than after alkaline
hydrolysis (137.13 mg GAE/kg fw). Therefore, the longer
incubation time of the gastric stage could lead to a higher
release of free phenolics, and the acidic medium and the
presence of digestive enzymes may also facilitate the release of
some bound phenolics.68
The sum of the OUT+IN portion represents the entire
intestinal phase. Compared to the gastric step of digestion,
after the intestinal stage (IN+OUT), the TPC value was only
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Table 4. Change in the Individual Phenolics and Anthocyanins of Fresh and Dried Plums during In Vitro Digestionab
oral gastric intestinal R% BI %
IN OUT
Phenolics (mg100/g dw)
gallic acid fresh 1.68 ± 0.00ab 2.20 ± 0.05a 0.95 ± 0.05b 2.39 ± 0.60a 8.65 ± 0.40B 30.85 ± 8.23B
HAD 0.80 ± 0.02b 1.68 ± 0.05a
0.28 ± 0.00c 1.67 ± 0.01a 4.63 ± 0.66C 32.02 ± 4.15B
VD 0.59 ± 0.01b 0.55 ± 0.00b
0.27 ± 0.00c 1.08 ± 0.06a 4.03 ± 0.01C 20.05 ± 0.99B
US-VD 0.88 ± 0.05b 1.24 ± 0.00a 0.43 ± 0.02c 1.33 ± 0.26a 24.64 ± 1.07A 100.92 ± 16.04A
FD 0.60 ± 0.02c 1.37 ± 0.03a
0.30 ± 0.05d 0.86 ± 0.07b 4.47 ± 0.72C 17.26 ± 1.54B
neochlorogenic acid fresh 3.03 ± 0.08b 77.13 ± 7.40a
0.04 ± 0.01b 0.57 ± 0.00b 0.04 ± 0.01A 0.65 ± 0.01D
HAD 7.03 ± 0.72b 24.17 ± 0.74a nd 1.35 ± 0.04c 2.49 ± 0.08C
VD 0.10 ± 0.01c 0.34 ± 0.04b nd 0.44 ± 0.02a 0.71 ± 0.02D
US-VD 0.18 ± 0.01c 0.55 ± 0.00b nd 0.81 ± 0.01a 54.89 ± 0.53A
FD 16.81 ± 1.42b 78.21 ± 0.68a nd 8.67 ± 1.27c 5.59 ± 0.82B
chlorogenic acid Fresh 5.65 ± 0.12b 41.24 ± 4.02a 1.65 ± 0.03bc 0.22 ± 0.01d 3.29 ± 0.44B 3.74 ± 0.50C
HAD 8.49 ± 0.68b 16.57 ± 0.98a 0.96 ± 0.10d 6.04 ± 0.74c 3.05 ± 0.82BC 22.20 ± 6.23B
VD 0.18 ± 0.00c 1.22 ± 0.08a
0.08 ± 0.01c 0.37 ± 0.03b 0.34 ± 0.08C 1.90 ± 0.02C
US-VD 0.19 ± 0.06c 4.07 ± 0.01a
0.08 ± 0.02d 0.35 ± 0.00b 8.76 ± 2.06A 45.58 ± 0.36A
FD 9.69 ± 0.42c 40.38 ± 2.17a 1.58 ± 0.12d 34.42 ± 2.30b 1.97 ± 0.27BC 45.02 ± 5.59A
caffeic acid fresh 1.11 ± 0.08b 2.54 ± 0.11a 0.55 ± 0.01d 0.87 ± 0.00c 7.84 ± 0.25B 20.29 ± 0.77C
HAD 1.29 ± 0.23b 2.59 ± 0.08a
0.17 ± 0.00d 0.58 ± 0.03c 3.66 ± 0.11B 16.18 ± 0.80C
VD 0.63 ± 0.13b 1.53 ± 0.05a
0.14 ± 0.00c 0.29 ± 0.00c 3.93 ± 0,01B 11.86 ± 0.13C
US-VD 0.41 ± 0.01b 1.26 ± 0.03a 0.23 ± 0.01b 0.44 ± 0.01b 25.15 ± 4.23A 73.05 ± 15.58A
FD 1.07 ± 0.04c 3.88 ± 0.07a 0.20 ± 0.01d 1.80 ± 0.01b 4.37 ± 0.18B 43.69 ± 2.07B
ellagic acid fresh 0.80 ± 0.80b 2.52 ± 0.07a
1.06 ± 0.16b 1.82 ± 0.04ab 8.04 ± 1.83A 21.81 ± 3.29B
HAD 0.73 ± 0.06b 2.80 ± 0.46a 0.37 ± 0.03b 0.50 ± 0.02b 5.42 ± 0.48B 12.66 ± 0.81C
VD 0.88 ± 0.00b 1.57 ± 0.03a nd 0.52 ± 0.01c 6.83 ± 0.07C
US-VD 0.87 ± 0.07b 1.19 ± 0.02a nd 0.94 ± 0.03b 59.99 ± 1.34A
FD 1.33 ± 0.01a 1.55 ± 0.05a 0.70 ± 0.01b 1.55 ± 0.38a 9.22 ± 0.43A 29.62 ± 5.93B
ferulic acid fresh 0.21 ± 0.00c 0.34 ± 0.01a 0.12 ± 0.00d 0.26 ± 0.00b 7.95 ± 0.07B 24.38 ± 0.54C
HAD 0.15 ± 0.04c 0.50 ± 0.00a 0.04 ± 0.00c 0.29 ± 0.08d 2.82 ± 0.42B 22.41 ± 6.56C
VD 0.08 ± 0.01b 0.29 ± 0.00a
0.03 ± 0.00c 0.10 ± 0.00b 3.88 ± 0.65B 15.95 ± 1.29C
US-VD 0.13 ± 0.01b 0.26 ± 0.09a
0.07 ± 0.00b 0.09 ± 0.00b 17.50 ± 4.33A 40.00 ± 4.33B
FD 0.15 ± 0.00c 0.74 ± 0.03b 0.03 ± 0.005d 1.18 ± 0.03a 4.44 ± 0.00B 135.55 ± 4.44A
quercetin fresh nd 3.85 ± 0.00a nd 3.32 ± 0.52a 11.45 ± 0.04A
HAD 1.05 ± 0.00a 1.05 ± 0.001a nd 1.05 ± 0.00a 6.62 ± 0.08B
VD 1.04 ± 0.00b 1.05 ± 0.00ab nd 1.06 ± 0.00a 6.65 ± 0.06B
US-VD nd nd nd
FD 1.06 ± 0.00ab 1.05 ± 0.001b 0.63 ± 0.00c 1.06 ± 0.00a 3.99 ± 0.00A 6.69 ± 0.13B
rutin fresh 0.45 ± 0.04b 14.90 ± 1.87a
0.64 ± 0.04b 0.86 ± 0.10b 8.93 ± 0.22A 20.96 ± 0.06C
HAD 1.78 ± 0.24c 4.87 ± 0.10a
0.22 ± 0.00d 2.77 ± 0.17b 6.45 ± 0.43B 87.58 ± 3.04AB
VD 1.42 ± 0.41b 3.13 ± 0.10a 0.04 ± 0.00c 0.18 ± 0.02c 0.83 ± 0.13D 4.10 ± 0.43C
US-VD 0.19 ± 0.07bc 2.53 ± 0.11a
0.04 ± 0.00c 0.31 ± 0.08b 10.43 ± 1.55A 76.36 ± 16.01B
FD 3.97 ± 0.01c 10.32 ± 0.24b
0.51 ± 0.03d 12.01 ± 1.16a 4.30 ± 0.12C 104.30 ± 0.12A
Anthocyanins (mg/100 g dw)
Cyn-3-O-glc fresh nd 17.76 ± 0.35 nd nd
HAD nd nd nd nd
VD nd 0.58 ± 0.02 nd nd
US-VD nd nd nd nd
FD nd 19.40 ± 1.96 nd nd
Pn-3̅-O-glc fresh nd 6.83 ± 1.25 nd nd
HAD nd nd nd nd
VD nd nd nd nd
US-VD nd nd nd nd
FD nd 6.25 ± 0.23 nd nd

a
Data are expressed as mean ± SD of triplicate measurements. Different lowercase letters in the same row for each sample are significantly different
(p < 0.05) among digestion phases. Different capital letters on the same column for RI% and BI% are significantly different (p < 0.05) among prune
samples. % (recovery percent) = CIN/Cnondigested × 100 BI%, (bioaccessibility index) = Cintestinal (IN+OUT)/Cnondigested cyn-3-O-glc: cyanidin 3-O-
glucoside, Pn3̅-O-glc: peonidin 3-O-glucoside. nd: not detected. bHAD, hot-air-drying; VD, vacuum-drying; US-VD, ultrasound-assisted vacuum-
drying; and FD, freeze-drying.

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increased in HAD and US-VD samples.25 The FC reagent of
the TPC assay could also give positive results with the Maillard
reaction products;69 therefore, due to the longer drying time,
the HAD and US-VD prunes could have Maillard reaction
products that could be available in the intestinal digestive
slurry and present higher TPC values. The bioaccessibility (BI,
%) of TPC in our plum samples ranged from 13.23% (VD) to
113.01% (US-VD) (Table 2). The BI (%) of TPC from
pomegranate arils was reported to change from 50.61 (fresh)
to 97.25% (US-VD).70 Compared to the gastric stage, there
was a significant decrease in the TFC and TA values in all
samples due to intestinal digestion conditions. The loss of TFC
and TA was associated with their instability under the alkaline
conditions of the small intestine, which could be related to the
oxidation and polymerization reactions they undergo, the
formation of higher-molecular-weight phenolic derivatives with
low solubility, and different chemical properties.24,29 The
bioaccessibility (%) of TFC was highest in FD (21.20%) and
US-VD (15.95%) and lowest in fresh (3.64%) samples (Table
2). Although the US-VD sample has the highest bioaccessi-
bility of TPC, TA, CUPRAC, and ABTS values, it is mostly
related to the lower initial level of the measured properties in
the extract and not because of the highest value determined
after digestion.
The antioxidant activity of the prune samples was examined
by DPPH, CUPRAC, ABTS, and FRAP assays. The alteration
in the antioxidant activity values of digested samples did not
exhibit a consistent pattern across all assays. As a parallel with
the TPC value, except for the DPPH assay, the antioxidant
activities increased after the gastric phase and decreased in the
intestinal phase. It could be related to the assay conditions;
only the DPPH assay has a radical soluble in the organic
solvent, whereas the other assays were all conducted in an
aqueous environment. As in our work, ABTS and hydroxyl
radical scavenging assays showed that the gastric digestion
products of mulberry fruits had the highest antioxidant activity
when compared to intestinal samples.71 ABTS radical
scavenging activities decreased by 36% and FRAP decreased
by 40% during the intestinal degradation of red cabbage. This
reduction in the parameters could be due to unidentified
chemical transformations of the phenolic compounds, in
particular, of the acylated anthocyanins.72
The residues of the samples after the small intestinal
digestion were extracted and analyzed the same way as for the
undigested samples. The TPC, TFC, TA, and antioxidant
activity values yielded by the digested residues were quite low
compared to those obtained before digestion (Table 3). The
recovery values of TPC in those digested residues were
changed from 1.18 to 14.50% in dried samples; the lowest was
determined in FD, and the highest was in HAD and VD
samples, while it was 3.47% in fresh plum. In the previous
studies conducted on grape samples, it was also indicated that
a significant portion of phenolics was extracted from the
digested fractions.25,44
3.6. Changes in the Levels of Individual Phenolics of
Fresh and Dried Plums during In Vitro Digestion. The
change of individual phenolics and anthocyanins (mg/100 g
dw) upon digestion is given in Table 4. As expected in all
samples, the amount of individual phenolics increased from the
oral to the gastric phase.73 Compared to the previous digestion
step, after intestinal digestion (IN+OUT), the amount of
neochlorogenic, chlorogenic, and caffeic acids decreased in all
samples, while the amount of other determined compounds
12720
Article
(gallic acid, ellagic acid, ferulic acid, and rutin) either
decreased or increased depending on the samples. Cyn-3-O-
glc and pn-3-O-glc were only detected in the gastric phase of
fresh, VD, and FD samples; no anthocyanins were recovered
after intestinal digestion. During the in vitro digestion of fresh
and sun-dried figs, all phenolic acids and anthocyanins
decreased by both drying and digestion.19 Anthocyanins
cannot maintain their stability due to the high pH when
passing through the gastrointestinal tract and the increase in
ambient temperature during drying processes.74 It was also
reported that the decrease in the bioaccessibility of
anthocyanins could be related to their transformation into
other phenolic substances during in vitro digestion, for
example, cyn-3-O-glc and cyn-3-O-rut could be converted to
cyanidin aglycones, ferulic acid, and caffeic acids.75
Both the amount of individual substances (Table 4) and
other measured properties (Table 2) passing into the IN
medium through the dialysis tube have generally been lower.
The possible formation of compounds with high molecular
weight in the intestinal phase due to polymerization reactions
could reduce the amount passing through to the IN phase.
Compared to the gastric stage, the loss of stability of phenolic
compounds in intestinal fluid, oxidation, and polymerization of
phenolic compounds at high pH could be the reason for the
reduced level of individual phenolics in the intestinal
phase.64,76 In the previous studies,29,30 the main phenolic
compounds determined in the bound fraction of plum fruit
were reported to be catechin, epicatechin, caffeic acid,
protocatechuic acid, ellagic acid, and ferulic acid. While Yu
et al.29 did not determine chlorogenic and neochlorogenic
acids, the main phenolic acids of plums, in the free phenolic
fraction, Seke et al.30 reported that the bound fraction has
chlorogenic acid content at a similar level to the free fraction.
During in vitro digestion conditions, the degradation and
polymerization of phenolics and their release from the matrix
would occur simultaneously. For example, from the gastric to
intestinal stage, the ferulic acid and ellagic acid contents of
fresh and FD prunes were increased, while their content in the
other samples was decreased (Table 4). The content of gallic
acid increased in all samples except FD prunes, whose
concentration did not change between the digestion steps.
The bioaccessibility of neochlorogenic, chlorogenic, and
caffeic acids in fresh plums were 0.65, 3.74, and 20.29%,
respectively. While Yu et al.29 determined the bioaccesibility of
chlorogenic acid in plums as 23.11%, Seke et al.30 could not
determine the chlorogenic acid in the digested plums. The
bioaccessibility of caffeic acid in plums was reported as 19.10%
by Seke et al.30 Considering both the amount of phenolics
reached in the small intestinal phase and the bioaccessibility
index (%) together, FD provided higher values compared to
those of fresh plum and other dried samples. Among dried
samples, considering the amount of those phenolics at the
intestinal stage, the highest concentration was detected in FD
prunes, followed by HAD samples. The bioaccessibility of
neochlorogenic, chlorogenic, and caffeic acids in FD prunes
was 5.59, 45.02, and 43.69% and 2.49, 22.20, and 16.18% in
HAD prunes, respectively (Table 4). Our results confirmed
that the structural changes that occurred during the drying
affected the release of phenolics from the food matrix.
Although the highest bioaccessibility was encountered for
US-VD prunes (such as 54.89% for neochlorogenic acid,
45.58% for chlorogenic acid, and 73.05% for caffeic acid), it
was due to their comparably lower initial values in the
https://doi.org/10.1021/acsomega.3c08383
ACS Omega 2024, 9, 12711−12724
ACS Omega http://pubs.acs.org/journal/acsodf Article

Figure 2. Heat maps representing the change of concentrations of individual phenolic compounds, TPC, TFC, TA, DPPH, CUPRAC, ABTS, and
FRAP in the oral, gastric, and intestinal phase of fresh [A], FD [B], VD [C], HAD [D], and US-VD [E] samples. HAD, hot-air-drying; VD,
vacuum-drying; US-VD, ultrasound-assisted vacuum-drying; and FD, freeze-drying. TPC; total phenolic content, TFC; total flavonoid content, TA;
total monomeric anthocyanins, DPPH: 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, CUPRAC: copper reducing antioxidant capacity,
ABTS: 2,2′-azino-bis (3-ethylbenzothiazoline6-sulfonic acid), and FRAP ferric reducing antioxidant power.

nondigested samples, not because of the amount available in
the intestinal stage.
Similar to our findings, it was 38.48% for fresh grapes and
increased to 110.18% by vacuum-drying.25 The level of
individual phenolics in the residues after digestion is given in
Table 3. Our results showed that all samples yielded a very low
content of individual phenolics in the free phenolic fraction of
the digested residue. Except for gallic and chlorogenic acids,
none of the individual phenolics were detected in the digested
residue of fresh plum. The ratio of the individual phenolics
measured in the residue to those of the undigested sample
ranged from 1.43 to 8.49% in the dried samples except US-VD
12721
prunes (14.89−31.03%) because of the lower initial level of
phenolics in the undigested sample. Although in vitro digestion
models were useful tools for assessing the proportion of food
constituents that were released from the matrix, which then
became available for absorption, they did not entirely replicate
the complex physicochemical and physiological processes that
occur in the human digestive tract.77
Bobrich et al.27 proposed that a meal of two large plums (∼
200 g) would easily provide up to 140 mg of bound phenolics,
and these plant matrix/cell wall-bound phenolics are trans-
ported to the colon without further degradation or absorption.
The polyphenols that cannot be extracted from the matrix in
https://doi.org/10.1021/acsomega.3c08383
ACS Omega 2024, 9, 12711−12724
ACS Omega http://pubs.acs.org/journal/acsodf
the stomach and small intestine travel through the gastro-
intestinal tract as insoluble substrates, reaching the colon,
where they release single polyphenols and different bioavailable
metabolites through the action of bacterial microbiota.78
Additionally, a heat map was created (Figure 2) to provide a
comprehensive visual representation of the change in
concentrations of individual phenolics, TPC, TFC, TA, and
antioxidant activities during each phase of in vitro digestion
(oral, gastric, and intestinal) for both fresh and dried plums.
Colors in the heat maps exhibited the intensity of the amount
of phenolic compounds and antioxidant activity during various
stages of in vitro digestion. The colors ranged from brown for
low levels of phenolic compounds and antioxidant activity to
green for high levels of phenolic compounds and antioxidant
activity. After oral digestion, the concentration of neo-
chlorogenic acid, which was represented by the green color,
was the highest in fresh, HAD, and FD samples. On the other
hand, the antioxidant activity (ABTS assay) of fresh and VD
samples was highest in the intestinal stage, as shown by the
green color.
4. CONCLUSIONS
In terms of the color, rehydration capacity, and retention of
initial phenolic compounds, the most favorable drying method
was freeze-drying (FD), followed by vacuum-drying (VD) and
hot-air-drying (HAD). Ultrasound-assisted vacuum-drying
(US-VD) was found to be the least desirable method for the
dehydration of plums. The morphology and structure of the
prunes differed depending on the drying method, which could
potentially influence how bioactive components were released
into the simulated digestion model. The changes in phenolic
compounds in plums varied throughout the digestion stages,
depending on the state of the food (fresh or dried) and the
drying method employed. Based on the bioaccessibility index
of measured properties (phenolics and antioxidant activities)
in digested samples, FD prunes yielded the highest values,
followed by HAD samples. In this study, only HPLC-DAD
could have been employed to measure the changes of
individual phenolics during digestion. Due to the complexity
of phenolic compounds and their possible transformation
products during gastrointestinal digestion, more comprehen-
sive determination methods such as LC-MS/MS could be
utilized for further research to identify the unknown
constituents by providing information about the structural
and molecular weight characterization. Further research should
also examine the changes in free and bound phenolics in plum
residues that remain after each digestion step, as well as the
effects of predrying treatment methods for removing the waxy
components of plums on the status of bioactive components
during in vitro digestion.
■ ASSOCIATED CONTENT
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsomega.3c08383.
The proximate composition of fresh plums (Table S1);
the change in color parameters of fresh and dried plums
(Table S2); and rehydration rates of dried plums at 25
°C (A) and 50 °C (B): FD, freeze-drying; VD, vacuum-
drying; HAD, hot-air-drying; US-VD, ultrasound-assis-
ted vacuum-drying (Figure S1) (PDF)
12722
Article
■ AUTHOR INFORMATION
Corresponding Author
Ayse Karadag − Department of Food Engineering, Faculty of
Chemical and Metallurgical Engineering, Yildiz Technical
University, 34210 Istanbul, Turkey; orcid.org/0000-
0001-8615-7321; Email: karadaga@yildiz.edu.tr; Fax: +90
212 383 4571
Authors
Elif Yener − Department of Food Engineering, Faculty of
Chemical and Metallurgical Engineering, Yildiz Technical
University, 34210 Istanbul, Turkey; Food Institute,
TUBITAK Marmara Research Center, Gebze 41470, Turkey
Oznur Saroglu − Department of Food Engineering, Faculty of
Chemical and Metallurgical Engineering, Yildiz Technical
University, 34210 Istanbul, Turkey
Osman Sagdic − Department of Food Engineering, Faculty of
Chemical and Metallurgical Engineering, Yildiz Technical
University, 34210 Istanbul, Turkey
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsomega.3c08383
Notes
The authors declare no competing financial interest.
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https://doi.org/10.1021/acsomega.3c08383
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