Industrial Crops & Products 131 (2019) 62–69

Contents lists available at ScienceDirect

Industrial Crops & Products

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

Influence of in vitro human digestion on the bioavailability of phenolic T

content and antioxidant activity of Viburnum opulus L. (European cranberry)
fruit extracts
⁎
Timur Hakan Barak, Engin Celep , Yiğit İnan, Erdem Yesilada
Yeditepe University, Faculty of Pharmacy, Department of Pharmacognosy, 34755, Istanbul, Turkey

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

Keywords: Although there is a wide array of antioxidant studies and related phenolic antioxidants on plants, bioavailability
Viburnum opulus phenomenon is often neglected. Design of this study based on evaluation of difference in antioxidant potential
In vitro digestion and phenolic profile of V. opulus fruits before and after in vitro gastrointestinal human digestion. For this purpose
HPTLC two extracts (methanolic and aqueous) were prepared from its fruits. After the digestion simulation procedure,
Antioxidant properties
total phenolic, phenolic acid, flavonoid contents were determined for all phases of digestion. High performance
Bioavailability
Chlorogenic acid
thin layer chromatography (HPTLC) was used for measurement of chlorogenic acid, major bioactive component
of the extracts. Bioavailability index was calculated for all phenolic content assays and for the bioactive com-
pound for accurate determination of alterations in phenolic profile via in vitro human digestion. For revelation of
the precise antioxidant potential of fruit extracts, a couple of free radical scavenging (DPPH and DMPD) and
metal reducing potential (CUPRAC and FRAP) assays and in addition a total antioxidant capacity assay were
conducted on all phases. The results showed that the methanolic extract is superior to aqueous counterpart in
terms of phenolic profile and antioxidant properties. Also, the major metabolite was negatively influenced by in
vitro simulation of human digestion.

1. Introduction
There are two hundred thirty Viburnum species (Adoxaceae)
showing a widespread natural distribution in the world as well as cul-
tivated ornamentally (Kraujalyte et al., 2013). In the flora of Turkey,
four Viburnum species are recorded: V. opulus L., V. orientale Pallas, V.
lantana L., and V. tinus L. (Altun et al., 2008). V. opulus (European
cranberrybush) is locally called as Gilaburu and its fruits have been
used for its healing properties in Turkey.
As a general approach its fruits are not preferred for consumption as
food due to its acidic taste, undesirable flavor and presence of some
unpleasantly smelling constituents (Kraujalis et al., 2017). However,
due to its reputation on health promoting properties, recently com-
mercial products prepared from the fruit juice collects growing demand
each year and that leads to the industrial importance of V. opulus fruits
(Ulger et al., 2012)
In Turkish folk medicine, V. opulus fruits particularly have a pro-
minence against kidney problems such as against nephralgia and to pass
kidney stone/sand (Altundag and Ozturk, 2011; Ilhan et al., 2014).
Meanwhile, it is also recorded that fruits are used as hypoglycemic and
to ease cough in Anatolian folk medicine (Fujita et al., 1995). Based
upon its reputation in folk medicine, a good number of pharmacological
activity studies have been conducted on V. opulus fruits. Antibacterial
(Sagdic et al., 2006) and antioxidant activities (Ersoy et al., 2017) have
been evaluated by in vitro studies. Its activity on alleviation of testicle
and sperm damages (Sarıözkan et al., 2017), antiurolithiatic effect
(Ilhan et al., 2014), protection against oxidative stress induces by
ischemia/reperfusion (Eken et al., 2017) have been studied in rat
models in vivo.
There is a growing interest on antioxidants and their health bene-
ficial effects. It is revealed that oxidative stress has a critical role on
numerous diseases such as cancer, diabetes, cardiovascular diseases etc.
(Celep et al., 2013). Daily intake of antioxidants has critical importance
in prophylaxis of these diseases. It is demonstrated that fruits are very
important antioxidant resources owing to their natural components
especially phenolic compounds (Rice-Evans et al., 1997). There are a
great number of in vitro studies reporting the antioxidant effects of
plants containing phenolic compounds. However, bioavailability of
compounds with antioxidant properties should also be examined so that
they can be demonstrated as beneficial to human health when these

⁎
Corresponding author.
E-mail address: ecelep@yeditepe.edu.tr (E. Celep).

https://doi.org/10.1016/j.indcrop.2019.01.037
Received 19 September 2018; Received in revised form 17 January 2019; Accepted 21 January 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
T.H. Barak et al.
mixture. The mixtures were incubated for 60 min. at room temperature.
Absorbance was calculated at 450 nm. Ascorbic acid used for calibra-
tion curve and as a reference substance.
2.7.2. Ferric reducing antioxidant power (FRAP)
Frap activity was estimated by the assay, described by Benzie and
Strain (1996). Freshly prepared FRAP reagent and Aliquots of the
samples were mixed and then diluted to 0.3 mL by distilled water and
then incubated at room temperature for 30 min. After incubation the
absorbance at 593 nm was measured. Ferrous chloride was used for
preparation of calibration curve. The results were expressed as mM
FeSO4 and BHT was used as a reference.
2.8. Determination of total antioxidant capacity
The method described by Prieto et al., (1999) was carried out for the
estimation of total antioxidant capacity. Aliquots of sample solutions
were added to the reaction mixture composed of sodium phosphate
monobasic, sulfuric acid and ammonium molybdate. Later, the mixtures
were incubated for 1.5 h at 95 °C at water bath. At the end of the in-
cubation process, the absorbance was measured at 695 nm. Ascorbic
acid was used for calibration curve and as standard substance
2.9. Statistics
Each of the tests and analyses were conducted in triplicate. After the
calculation of mean ± standard deviation, the results were statistically
compared with ANOVA test. Tukey-Kramer post hoc test was run for
multiple comparisons. p < 0.05 was defined as statistically significant
difference.
3. Results
3.1. Quantification of the phenolic profile of the samples
Data presented in Table 1 demonstrated total amounts of phenolic
compounds before and after the simulation of human digestion.
Amount of total phenolic compounds (TPC) was dramatically decreased
after the digestion procedure. TPC of VOM was decreased from
40.17 ± 1.11 mg/g GAE to 14.77 ± 0.18 and TPC of VOA decreased
from 25.64 ± 1.06 to 15.23 ± 0.31 in ND fraction and IN fraction,
respectively. Also total phenolic acids (TPA) in VOM were strikingly
reduced, the while reduction in VOA is modest. TPA of VOM was de-
creased from 50.98 ± 1.46 mg/g CAE to 36.87 ± 2.21 and TPA of
VOA was decreased 42.54 ± 1.13 to 38.56 ± 0.26 in ND fraction and
IN fraction, respectively. No extreme alterations were measured in both
extracts in total flavonoid content assay. Total flavonoid content was
Table 1
Spectrophotometric determination of phenolic profile of the extracts from V. opulus fruit and their bioavailability indexes.
Samples NDA PG
B,C a* b c
VOM-TPC 40.17 ± 1.11 26.43 ± 0.91
VOA-TPC 25.64a ± 1.06 21.08b ± 0.81
VOM-TPACD 50.98a ± 1.46 44.24b ± 3.41
VOA-TPAC 42.54a ± 1.13 40.86ab ± 0.46
VOM-TFCE 25.09a ± 0.77 25.61a ± 0.37
VOA-TFC 24.57a ± 0.47 24.18a ± 0.55
VOM: Methanolic extract of Viburnum opulus L.fruit.
VOA: Aqueous extract of Viburnum opulus L. fruits.
A
The abbreviations for samples are ND: non-digested, PG: postgastric, OUT: colon-available IN: bioavailable, BAvI: Bioavailability index.
B
The abbreviations for the analysis are TPC: Total phenolic content, TPAC: Total phenolic acid content, TFC: Total flavonoid content.
C
Results were expressed as the mean of triplicates ± standard deviation (S.D.) and as mg gallic acid equivalents (GAE) in 1 g sample.
D
Results were expressed as the mean of triplicates ± standard deviation (S.D.) and as mg caffeic acid equivalents (CAE) in 1 g sample.
E
Results were expressed as the mean of triplicates ± standard deviation (S.D.) and as mg quercetin equivalents (QE) in 1 g sample.
* Different letters in the same row indicate significance (p < 0.05).
65
Industrial Crops & Products 131 (2019) 62–69
slightly decreased in VOM from 25.09 mg/g QE ± 0.77 to
23.35 ± 0.37 and in VOA from 24.57 ± 0.47 to 22.02 ± 0.44.
Chlorogenic acid (Fig. 2) quantity of non-digested VOM extract, as
presented in Table 2, was determined as 34.42 ± 1.22 mg/g dry ex-
tract, which is higher than non-digested VOA extract, 26.76 ± 0.91.
Both extracts were demonstrated decline in post-gastric phase
(25.50 ± 0.45, 18.61 ± 0.07 respectively). Even though modest en-
hancement was measured in colon-available phase (31.56 ± 1.00 and
21.91 ± 0.43, respectively), notable diminishment was detected in
serum available phase (15.09 ± 0.32 and 13.39 ± 0.74, respectively).
Total bioavailability of the active metabolite in VOM extract was
measured as 38.90% in the meantime in VOA extract total bioavail-
ability was measured 56.40%.
3.2. Estimation of metal reducing capacity
Two different metal reducing assays were performed to assert fac-
tual results. As shown in Table 3, in CUPRAC assay, non-digested VOM
showed higher activity than non-digested VOA (208.87 ± 9.32 mg/g
AAE and 156.49 ± 4.32, respectively). After the gastric phase, cupric
reducing capacity of both extracts were reduced significantly
(156.36 ± 3.00 and 107.85 ± 11.48, respectively). Even though
colon-available (OUT) fractions have similar activity with post-gastric
(PG) fractions (164.01 ± 4.75, 100.93 ± 2.03 respectively), cupric
reducing capacity of both extract were diminished significantly in the
serum available phase (IN) (96.50 ± 1.68, 90.72 ± 2.82, respec-
tively).
In FRAP assay, both extracts showed similar activities for all phases.
Non-digested VOM and VOA displayed similar ferric reducing activity
(0.46 ± 0.05 mm/g FeSO4 equivalent, 0.41 ± 0.09 respectively).
Downtrend of reducing activity was analogous in both extracts. Post-
gastric activities of VOM and VOA were not significantly different with
non-digested phases (0.45 ± 0.12 and 0.40 ± 0.01, respectively).
Likewise, colon-available phases of both extracts exhibited parallel
activity with non-digested and post-gastric phases (0.36 ± 0.01,
0.38 ± 0.03 respectively). Serum-available phases of both extracts
indicated slight but significant decline, ultimately both of them showed
similar activity (0.29 ± 0.04, 0.28 ± 0.02, respectively).
3.3. Determination of free radical scavenging potential
Two different free radical scavenging activity assays were operated
to obtain higher accuracy rates. In DMPD (Dimethyl-4-phenylenedia-
mine) assay, both extracts showed no significant changes for all phases.
Non-digested, post-gastric, colon available and serum-available phases
possessed resembling DMPD radical activity. Results were demon-
strated in Table 3.
OUT IN BAvI (%)
d
23.77 ± 0.51 14.77 ± 0.18 36.77 %
19.28c ± 0.04 15.23d ± 0.31 59.40 %
43.75b ± 0.96 36.87c ± 2.21 72.32 %
40.28b ± 0.98 38.56bc ± 0.26 90.64 %
25.54a ± 0.88 23.35b ± 0.37 93.06 %
24.57a ± 0.41 23.02b ± 0.44 93.69 %
T.H. Barak et al. Industrial Crops & Products 131 (2019) 62–69

Fig. 2. A) HPTLC chromatograms of: 1. VOM ND, 2. VOM PG, 3. Chlorogenic acid (Rf: 030), 4. VOM OUT, 5. VOM IN, 6. Chlorogenic acid, 7. VOA ND, 8. VOA PG,
9.Chlorogenic acid, 10. VOA OUT, 11. VOA IN. Mobile phase: AcOEt/ CHCl2/ CH3COOH/ HCOOOH/ H2O (100:25:10:10:11); Derivatization: NPR reagent.
Visualization: 366 nm.
B) UV spectra of all tracks at 366 nm.

Table 2 than post-gastric and colon-available phase (PG: 40.69 ± 0.48, OUT:
Quantification of the major bioactive compound by HPTLC and its bioavail- 36.73 ± 2.14, IN: 30.72 ± 2.89), while VOA extract showed no sig-
ability index. nificant changes after post-gastric phase (PG: 33.09 ± 0.82, OUT:
Samples NDA PG OUT IN BAvI (%) 31.35 ± 1.37. IN: 30.40 ± 1.93)

a* b c d
VOM- Chlorogenic 34.42 25.50 31.56 13.39 38.90 %
acidB ± 1.22 ± 0.45 ± 1.00 ± 0.74 4. Discussion
VOA- Chlorogenic 26.76a ± 18.61b 21.91c 15.09d 56.40 %
acid 0.91 ± 0.07 ± 0.43 ± 0.32 Recent medicinal approach asserts that natural and biologically
active compounds originated from plants have prior preference for
VOM: Methanolic extract of Viburnum opulus L. fruit.
VOA: Aqueous extract of Viburnum opulus L. fruits.
prophylaxis and therapy due to their low side effect profile and better
A
The abbreviations for samples are ND: non-digested, PG: postgastric, OUT: patient compliance compared to synthetic chemical pharmaceuticals.
colon-available, IN: bioavailable, BAvI: Bioavailability index. Recent investigations have stated expressly the role of oxidative stress
B
Results are expressed as mg/g dry extract with standart deviations. in the etiology of many health problems particularly of chronic dis-
* Different letters in the same row indicate significance (p < 0.05). orders and therefore antioxidant activity of plants or phytochemicals
has become highly important (Wilcox, 2005). These chronic diseases
In DPPH (1,1-diphenyl-2-picrylhydrazyl) assay, post-gastric phases such as Alzheimer’s, diabetes, cancer etc. do not have a specific cure and
of both extracts had negligible changes with non-digested phase. In require lifelong symptomatic treatment; thereby prevention from these
addition, both extracts demonstrated alleviation in small quantities in diseases economizes high costs. Phenolic compounds including phe-
DPPH radical scavenging activity at serum available fraction (Table 3). nolic acids, flavonoids, anthocyanidins, etc. are among the naturally
occurring antioxidants and fruits are generally valuable resources of
such phenolic compounds. Supplementing diet with fruits with ad-
3.4. Measurement of total antioxidant capacity vanced antioxidant properties is beneficial for overall health and aug-
ments the possibility of prevention of chronic diseases. A previous re-
Total antioxidant capacity assay was operated as an alternative port demonstrated that 1 g of daily consumption of phenolic
method for the determination of precise antioxidant activity of extracts. compounds through enriched diet by fresh vegetables and fruits may
Results were given in Table 3. In TOAC assay VOM extract demon- help inhibiting mutagenesis and carcinogenesis (Tanaka et al., 2003).
strated higher activity than VOA extracts (56.89 ± 5.14 mg/g AAE, Fruits and other plant parts consumed as food resources have been
49.07 ± 6.20 respectively). After gastric simulation phase activity of investigated numerous times. On the other hand, bioavailability of the
both extracts dropped significantly (40.69 ± 0.48 and 33.09 ± 0.82, compounds responsible for the activity is generally neglected in the vast
respectively). In VOM extract, serum-available phase had lesser activity majority of the studies. Yet, it is indispensable to determine the

66
T.H. Barak et al.
mixture. The mixtures were incubated for 60 min. at room temperature.
Absorbance was calculated at 450 nm. Ascorbic acid used for calibra-
tion curve and as a reference substance.
2.7.2. Ferric reducing antioxidant power (FRAP)
Frap activity was estimated by the assay, described by Benzie and
Strain (1996). Freshly prepared FRAP reagent and Aliquots of the
samples were mixed and then diluted to 0.3 mL by distilled water and
then incubated at room temperature for 30 min. After incubation the
absorbance at 593 nm was measured. Ferrous chloride was used for
preparation of calibration curve. The results were expressed as mM
FeSO4 and BHT was used as a reference.
2.8. Determination of total antioxidant capacity
The method described by Prieto et al., (1999) was carried out for the
estimation of total antioxidant capacity. Aliquots of sample solutions
were added to the reaction mixture composed of sodium phosphate
monobasic, sulfuric acid and ammonium molybdate. Later, the mixtures
were incubated for 1.5 h at 95 °C at water bath. At the end of the in-
cubation process, the absorbance was measured at 695 nm. Ascorbic
acid was used for calibration curve and as standard substance
2.9. Statistics
Each of the tests and analyses were conducted in triplicate. After the
calculation of mean ± standard deviation, the results were statistically
compared with ANOVA test. Tukey-Kramer post hoc test was run for
multiple comparisons. p < 0.05 was defined as statistically significant
difference.
3. Results
3.1. Quantification of the phenolic profile of the samples
Data presented in Table 1 demonstrated total amounts of phenolic
compounds before and after the simulation of human digestion.
Amount of total phenolic compounds (TPC) was dramatically decreased
after the digestion procedure. TPC of VOM was decreased from
40.17 ± 1.11 mg/g GAE to 14.77 ± 0.18 and TPC of VOA decreased
from 25.64 ± 1.06 to 15.23 ± 0.31 in ND fraction and IN fraction,
respectively. Also total phenolic acids (TPA) in VOM were strikingly
reduced, the while reduction in VOA is modest. TPA of VOM was de-
creased from 50.98 ± 1.46 mg/g CAE to 36.87 ± 2.21 and TPA of
VOA was decreased 42.54 ± 1.13 to 38.56 ± 0.26 in ND fraction and
IN fraction, respectively. No extreme alterations were measured in both
extracts in total flavonoid content assay. Total flavonoid content was
Table 1
Spectrophotometric determination of phenolic profile of the extracts from V. opulus fruit and their bioavailability indexes.
Samples NDA PG
B,C a* b c
VOM-TPC 40.17 ± 1.11 26.43 ± 0.91
VOA-TPC 25.64a ± 1.06 21.08b ± 0.81
VOM-TPACD 50.98a ± 1.46 44.24b ± 3.41
VOA-TPAC 42.54a ± 1.13 40.86ab ± 0.46
VOM-TFCE 25.09a ± 0.77 25.61a ± 0.37
VOA-TFC 24.57a ± 0.47 24.18a ± 0.55
VOM: Methanolic extract of Viburnum opulus L.fruit.
VOA: Aqueous extract of Viburnum opulus L. fruits.
A
The abbreviations for samples are ND: non-digested, PG: postgastric, OUT: colon-available IN: bioavailable, BAvI: Bioavailability index.
B
The abbreviations for the analysis are TPC: Total phenolic content, TPAC: Total phenolic acid content, TFC: Total flavonoid content.
C
Results were expressed as the mean of triplicates ± standard deviation (S.D.) and as mg gallic acid equivalents (GAE) in 1 g sample.
D
Results were expressed as the mean of triplicates ± standard deviation (S.D.) and as mg caffeic acid equivalents (CAE) in 1 g sample.
E
Results were expressed as the mean of triplicates ± standard deviation (S.D.) and as mg quercetin equivalents (QE) in 1 g sample.
* Different letters in the same row indicate significance (p < 0.05).
65
Industrial Crops & Products 131 (2019) 62–69
slightly decreased in VOM from 25.09 mg/g QE ± 0.77 to
23.35 ± 0.37 and in VOA from 24.57 ± 0.47 to 22.02 ± 0.44.
Chlorogenic acid (Fig. 2) quantity of non-digested VOM extract, as
presented in Table 2, was determined as 34.42 ± 1.22 mg/g dry ex-
tract, which is higher than non-digested VOA extract, 26.76 ± 0.91.
Both extracts were demonstrated decline in post-gastric phase
(25.50 ± 0.45, 18.61 ± 0.07 respectively). Even though modest en-
hancement was measured in colon-available phase (31.56 ± 1.00 and
21.91 ± 0.43, respectively), notable diminishment was detected in
serum available phase (15.09 ± 0.32 and 13.39 ± 0.74, respectively).
Total bioavailability of the active metabolite in VOM extract was
measured as 38.90% in the meantime in VOA extract total bioavail-
ability was measured 56.40%.
3.2. Estimation of metal reducing capacity
Two different metal reducing assays were performed to assert fac-
tual results. As shown in Table 3, in CUPRAC assay, non-digested VOM
showed higher activity than non-digested VOA (208.87 ± 9.32 mg/g
AAE and 156.49 ± 4.32, respectively). After the gastric phase, cupric
reducing capacity of both extracts were reduced significantly
(156.36 ± 3.00 and 107.85 ± 11.48, respectively). Even though
colon-available (OUT) fractions have similar activity with post-gastric
(PG) fractions (164.01 ± 4.75, 100.93 ± 2.03 respectively), cupric
reducing capacity of both extract were diminished significantly in the
serum available phase (IN) (96.50 ± 1.68, 90.72 ± 2.82, respec-
tively).
In FRAP assay, both extracts showed similar activities for all phases.
Non-digested VOM and VOA displayed similar ferric reducing activity
(0.46 ± 0.05 mm/g FeSO4 equivalent, 0.41 ± 0.09 respectively).
Downtrend of reducing activity was analogous in both extracts. Post-
gastric activities of VOM and VOA were not significantly different with
non-digested phases (0.45 ± 0.12 and 0.40 ± 0.01, respectively).
Likewise, colon-available phases of both extracts exhibited parallel
activity with non-digested and post-gastric phases (0.36 ± 0.01,
0.38 ± 0.03 respectively). Serum-available phases of both extracts
indicated slight but significant decline, ultimately both of them showed
similar activity (0.29 ± 0.04, 0.28 ± 0.02, respectively).
3.3. Determination of free radical scavenging potential
Two different free radical scavenging activity assays were operated
to obtain higher accuracy rates. In DMPD (Dimethyl-4-phenylenedia-
mine) assay, both extracts showed no significant changes for all phases.
Non-digested, post-gastric, colon available and serum-available phases
possessed resembling DMPD radical activity. Results were demon-
strated in Table 3.
OUT IN BAvI (%)
d
23.77 ± 0.51 14.77 ± 0.18 36.77 %
19.28c ± 0.04 15.23d ± 0.31 59.40 %
43.75b ± 0.96 36.87c ± 2.21 72.32 %
40.28b ± 0.98 38.56bc ± 0.26 90.64 %
25.54a ± 0.88 23.35b ± 0.37 93.06 %
24.57a ± 0.41 23.02b ± 0.44 93.69 %
T.H. Barak et al. Industrial Crops & Products 131 (2019) 62–69

Fig. 2. A) HPTLC chromatograms of: 1. VOM ND, 2. VOM PG, 3. Chlorogenic acid (Rf: 030), 4. VOM OUT, 5. VOM IN, 6. Chlorogenic acid, 7. VOA ND, 8. VOA PG,
9.Chlorogenic acid, 10. VOA OUT, 11. VOA IN. Mobile phase: AcOEt/ CHCl2/ CH3COOH/ HCOOOH/ H2O (100:25:10:10:11); Derivatization: NPR reagent.
Visualization: 366 nm.
B) UV spectra of all tracks at 366 nm.

Table 2 than post-gastric and colon-available phase (PG: 40.69 ± 0.48, OUT:
Quantification of the major bioactive compound by HPTLC and its bioavail- 36.73 ± 2.14, IN: 30.72 ± 2.89), while VOA extract showed no sig-
ability index. nificant changes after post-gastric phase (PG: 33.09 ± 0.82, OUT:
Samples NDA PG OUT IN BAvI (%) 31.35 ± 1.37. IN: 30.40 ± 1.93)

a* b c d
VOM- Chlorogenic 34.42 25.50 31.56 13.39 38.90 %
acidB ± 1.22 ± 0.45 ± 1.00 ± 0.74 4. Discussion
VOA- Chlorogenic 26.76a ± 18.61b 21.91c 15.09d 56.40 %
acid 0.91 ± 0.07 ± 0.43 ± 0.32 Recent medicinal approach asserts that natural and biologically
active compounds originated from plants have prior preference for
VOM: Methanolic extract of Viburnum opulus L. fruit.
VOA: Aqueous extract of Viburnum opulus L. fruits.
prophylaxis and therapy due to their low side effect profile and better
A
The abbreviations for samples are ND: non-digested, PG: postgastric, OUT: patient compliance compared to synthetic chemical pharmaceuticals.
colon-available, IN: bioavailable, BAvI: Bioavailability index. Recent investigations have stated expressly the role of oxidative stress
B
Results are expressed as mg/g dry extract with standart deviations. in the etiology of many health problems particularly of chronic dis-
* Different letters in the same row indicate significance (p < 0.05). orders and therefore antioxidant activity of plants or phytochemicals
has become highly important (Wilcox, 2005). These chronic diseases
In DPPH (1,1-diphenyl-2-picrylhydrazyl) assay, post-gastric phases such as Alzheimer’s, diabetes, cancer etc. do not have a specific cure and
of both extracts had negligible changes with non-digested phase. In require lifelong symptomatic treatment; thereby prevention from these
addition, both extracts demonstrated alleviation in small quantities in diseases economizes high costs. Phenolic compounds including phe-
DPPH radical scavenging activity at serum available fraction (Table 3). nolic acids, flavonoids, anthocyanidins, etc. are among the naturally
occurring antioxidants and fruits are generally valuable resources of
such phenolic compounds. Supplementing diet with fruits with ad-
3.4. Measurement of total antioxidant capacity vanced antioxidant properties is beneficial for overall health and aug-
ments the possibility of prevention of chronic diseases. A previous re-
Total antioxidant capacity assay was operated as an alternative port demonstrated that 1 g of daily consumption of phenolic
method for the determination of precise antioxidant activity of extracts. compounds through enriched diet by fresh vegetables and fruits may
Results were given in Table 3. In TOAC assay VOM extract demon- help inhibiting mutagenesis and carcinogenesis (Tanaka et al., 2003).
strated higher activity than VOA extracts (56.89 ± 5.14 mg/g AAE, Fruits and other plant parts consumed as food resources have been
49.07 ± 6.20 respectively). After gastric simulation phase activity of investigated numerous times. On the other hand, bioavailability of the
both extracts dropped significantly (40.69 ± 0.48 and 33.09 ± 0.82, compounds responsible for the activity is generally neglected in the vast
respectively). In VOM extract, serum-available phase had lesser activity majority of the studies. Yet, it is indispensable to determine the

66
T.H. Barak et al. Industrial Crops & Products 131 (2019) 62–69

Table 3
In vitro antioxidant activity of VOM* and VOA** before and after simulated human digestion.
A
Name of the analysis ND PG OUT IN

a *** a b
VOM-DPPH 103.59 ± 5.26 106.02 ± 2.79 92.28 ± 0.96 88.60b ± 3.18
scavenging act. B,#
VOA-DPPH 96.74a ± 4.15 92.88ab ± 4.75 93.41ab ± 2.11 87.22b ± 1.09
scavenging act.
VOM-DMPD 52.55a ± 4.87 46.41a ± 9.27 47.64a ± 7.67 42.11a ± 1.15
scavenging act. C,§
VOA- DMPD scavenging act. 55.00a ± 2.81 45.39a ± 6.64 49.48a ± 2.62 52.55a ± 6.47
VOM-FRAP D,## 0.46a ± 0.05 0.45a ± 0.08 0.36ab ± 0.01 0.29b ± 0.04
VOA-FRAP 0.41a ± 0.09 0.40ab ± 0.01 0.38ab ± 0.03 0.28b ± 0.02
VOM-CUPRAC E,§§ 208.87a ± 9.32 156.36b ± 3.00 164.01b ± 4.75 96.50c ± 1.68
VOA-CUPRAC 156.49a ± 4.32 107.85b ± 11.48 100.93bc ± 2.03 90.72c ± 2.82
VOM-Total antioxidant capacityE 56.89a ± 5.14 40.69b ± 0.48 36.73bc ± 2.14 30.72c ± 2.89
VOA- Total antioxidant capacity 49.07a ± 6.20 33.09b ± 0.82 31.35b ± 1.37 30.40b ± 1.93

P.S. FRAP activity of the reference compound “butylated hydroxytoluene (BHT)” is found to be 4.18 ± 0.26 mM FeSO4 eq. in 1 g sample.
A
The abbreviations for samples are ND: non-digested, PG: postgastric, OUT:colon-available, IN: bioavailable.
B
Results were expressed as the mean of triplicates ± standard deviation (S.D.) and as mg butylated hydroxyl toluene equivalents (BHTE) in 1 g sample.
C
Results were expressed as the mean of triplicates ± standard deviation (S.D.) and as mg Trolox equivalents (TE) in 1 g sample.
D
Results were expressed as the mean of triplicates ± standard deviation (S.D.) and as Mm FeSO4 equivalents in 1 g sample.
E
Results were expressed as the mean of triplicates ± standard deviation (S.D.) and as mg ascorbic acid equivalents (AAE) in 1 g sample.
* VOM: methanolic extract of Viburnum opulus L. fruits.
** VOA: aqueous extract of Viburnum opulus L. fruits.
*** Different letters in the same row indicate significance (p < 0.05).
#
2,2-diphenyl-1-picrylhydrazyl.
§
N,N-dimethyl-p-phenylendiamine.
##
Ferric reducing antioxidant power.
§§
Cupric reducing antioxidant capacity.

bioavailability of the active metabolites. When consumed orally plants
they are subjected to gastrointestinal tract conditions. These conditions
may highly affect the chemical structure and the bioavailability of the
plant metabolites as a consequence of variance in pH values, presence
of a variety of enzymes, body heat and other physical and biochemical
aspects. Also it is important that the active metabolite to be absorbable
into systemic circulation. It is essential for bioactive compounds to
reach sufficient concentration in target tissues to induce their biological
activity. Therefore, in this study a human digestion simulation was
conducted to investigate the antioxidant potential of V. opulus fruits
with a high degree of precision. Even though in vivo studies have higher
levels of certainty, ethical considerations and high economic and time
cost makes such in vitro studies more preferable (Mcdougall et al.,
2005). Moreover, correlation between in vitro simulation studies and in
vivo studies were found highly sufficient (Alminger et al., 2014).
Other research groups previously investigated antioxidant proper-
ties of V. opulus fruits. However, these studies overlooked the effects of
gastrointestinal system on extracts and the bioavailability of biologi-
cally active major metabolite. According to a study conducted on six
different genotypes of V. opulus fruit parts from Lithuania, total phe-
nolic contents varied from 5.47 ± 0.24 to 10.61 ± 0.42 mg GAE/g
dry extract (Kraujalyte et al., 2013). Total phenolic content of metha-
nolic extract of V. opulus fruits collected from Kayseri was calculated as
67.73 mg GAE/g by Eken et al., (2017). Rop et al., (2010) measured
total phenolic content and total flavonoid content of different V. opulus
cultivars harvested in different years. Results varied 6.80 ± 0.15 to
8.31 ± 0.21 mg GAE/g and 3.14 ± 0.17 to 4.89 ± 0.17 mg/g rutin
equivalent in total phenolic and flavonoid contents of fresh mass, re-
spectively. Ersoy et al., (2017) investigated both total phenolic and
flavonoid content of ten different genotypes of V. opulus fruits collected
from Sivas province, Turkey. Results varied from 621 ± 15 to
987 ± 32 mg GAE/100 g fruit weight and 202 ± 12 to 318 ± 16 mg
rutin equivalent/100 g fruits weight, respectively. However, total phe-
nolic acid content assay has not been conducted on V. opulus fruits
before to our knowledge. Although various research groups studied the
phenolic profile of V. opulus, phenolic bioavailability has not been taken
into consideration before. In this study, bioavailability of the phenolics
and their effects on antioxidant capacity were revealed. Results in
Table 1 demonstrated that all IN fractions possessed the lowest phenolic
profile. Total phenolic and phenolic acid contents of both extracts de-
creased significantly at IN fraction except total flavonoid content,
which showed no significant modification. Alterations in phenolic acid
and total phenolic amounts were more substantial than total flavonoid
content; since V. opulus fruits are meager in flavonoids wherefore its
biological activities might depend majorly on its total phenolics and
phenolic acids. These results imply that phenolic structures in the V.
opulus extracts have low stability in the GI tract. Reduction of the
phenolic compounds seems to be reasoned by some physical properties
of the GI tract like significant pH changes, body heat. Especially, al-
kaline medium of the small intestines is a major parameter of descent of
phenolic compounds (Chen et al., 2014). Another element that may
cause reduction of the phenolics is excessive enzyme activity in the
intestines. Enzymes may accelerate the hydrolyzation of the compounds
after gastric digestion phase. Previous reports revealed opposite out-
comes such as some studies showed escalation in the amount of phe-
nolic contents in contrast other studies demonstrated descent (Chen
et al., 2014; Bhatt and Patel, 2013; Baker et al., 2013; Celep et al.,
2017). It might be hypothesized that these variations in the results of
various studies originate from unique characteristic of the investigated
samples.
Chlorogenic acid is known as the major metabolite of V. opulus fruits
(Karaçelik et al., 2015). Perova et al. (2014) measured the amount of
chlorogenic acid in eleven different fruit samples collected from Russia.
Results were varied from 250 to 580 mg chlorogenic acid per 100 g
fresh fruits. Velioglu et al. (2006) investigated phenolic composition of
V. opulus fruits and showed that 54% of all phenolic components was
chlorogenic acid, a far more dominant metabolite than other phenolic
compounds. However, the bioavailability of chlorogenic acid of V.
opulus was not investigated before. Therefore, in this study the amount
of chlorogenic acid was measured by HPTLC in both extracts and in all
phases of simulated human digestion for ascertainment. Descending
trend in chlorogenic acid amount was consistent with total phenolic
acid and total phenolics assays. Descent in chlorogenic acid level sub-
sequent to digestion was reported previously by other studies (Vallejo

67
T.H. Barak et al.
et al., 2004; Gumienna et al., 2011). Besides, methanolic extract
showed higher phenolic profile in all assays (total phenolics, total
phenolic acids, total flavonoids and HPTLC analysis). For all assays,
bioavailability index rate was calculated as higher in aqueous extract
(Table 1 and 2). After the digestion procedure, bioavailable amount of
these compounds was measured as the same. Therefore, it can be
claimed that both extracts had a similar bioavailability trend. Simulated
human digestion method used in this study excluded gastric absorption
of polyphenolic compounds from extracts. A previous study reveals that
chlorogenic acid is rapidly absorbed from stomach of rats in its intact
form (Lafay et al., 2006). Another report indicates that chlorogenic acid
is highly bioavailable in humans (Farah et al., 2008). Based on this
information, it can be speculated that post-gastric substantiality of
chlorogenic acid enhances the biological activity of methanolic extract
and commercial fruit juice of V. opulus. Its biological activity is due to
the major metabolite, chlorogenic acid, which was measured in higher
amount in post-gastric phase than serum-available phase.
Antioxidant effect of the plant parts originates from diversified
mechanisms. To achieve an improved perspective, in this study couple
of metal reducing, free radical scavenging activity assays and ad-
ditionally a total antioxidant capacity assay were performed. Metal
reducing activity was studied with two different assays. CUPRAC assay
was studied for the first time on V. opulus fruits to our knowledge. In a
previous study about ferric reducing antioxidant power of V. opulus,
results varied from 55.77 ± 1.77 to 109.76 ± 1.37 Fe2+ μmol/g
(Kraujalyte et al., 2013). But that study ignored the influence of gas-
trointestinal tract on the bioactivity. Results of CUPRAC and FRAP as-
says were demonstrated in Table 3. Both extracts showed coherent
gradient with their phenolic profile and major metabolite in metal re-
ducing activity. Sagdic et al. (2006) measured total antioxidant capa-
city of methanolic fruit extracts of V. opulus via phosphomolybdenum
complex method before, still disregarding the effects of digestion
(315.50 ± 8.2 mg/g AAE). Likewise, total antioxidant activity reduced
at IN fraction just as total phenolics and metal reducing capacity. Pre-
vious reports affirmed similar outcomes on other fruit samples. In brief,
Bouayed et al. (2012) reported reduction of total phenolics at IN frac-
tion conduced to decline in ferric reducing activity. Our research group
also reported reduction in cupric ion reducing capacity and total anti-
oxidant capacity corresponds with alleviation on phenolic content in
fruit wines (Celep et al., 2015).
Free radical scavenging activity assays are prevalently used for the
definition of antioxidant properties of plant parts (Celep et al., 2018). In
this study DPPH and DMPD radical scavenging activity assays were
conducted. In a previous study Rop et al. (2010) measured DPPH
scavenging activity of V. opulus fruits from different cultivars and found
values up to 9.79 ± 0.14 g ascorbic acid equivalent/kg fresh mass. In
the present study DPPH assay both extracts showed slight but sig-
nificant changes when compared to non-digested and serum available
fractions. As observed in the metal reducing and total antioxidant as-
says, methanolic extract showed higher activity in DPPH assay, and
both extracts had similar activity at IN fraction. On the other and,
DMPD scavenging activity of V. opulus fruits was not studied before to
our knowledge. In DMPD assay, contrary to other assays, both extracts
had similar scavenging activity in all phases of digestion. When com-
pared to metal reducing, total antioxidant and total phenolic content
results, free radical scavenging activity either remained the same or
decreasing slightly after the digestion simulation.
This study takes account of exclusively phenolic compounds, for
that reason possible effects of non-phenolic compounds are in-
computable. In a similar manner Pavan et al. (2014) reported similarity
in antioxidant activity of digested papaya against undigested phase.
That study hypothesized that non-phenolic compounds possibly affect
antioxidant activity of plant sample, thus decline in phenolic amount is
compensated on in vitro bioactivity. Variance in the free radical
scavenging activity was also similar to total flavonoid content. It is also
possible that flavonoids are the major phenolic compounds of the
68
Industrial Crops & Products 131 (2019) 62–69
extract which are responsible for the free radical scavenging activity
rather than other phenolics.
In conclusion, this study demonstrated first time the antioxidant
properties of V. opulus fruits, considering bioavailability and influence
of GI tract on the activity profile of extracts. Experimental results have
clearly shown that phenolic contents and antioxidant properties of
fruits were influenced by digestion significantly. Moreover, active me-
tabolite of the fruit material showed decline after encountering with GI
tract conditions. In addition, absorption from colon to serum from
dialysis tube reduced availability of active metabolites significantly.
References
Alminger, M., Aura, A., Bohn, T., Dufour, C., El, S.N., Gomes, A., Karakaya, S., Mart, M.C.,
Mcdougall, G.J., Requena, T., Santos, C.N., 2014. In vitro models for studying sec-
ondary plant metabolite digestion and bioaccessibility. Compr. Rev. Food Sci. Food
Saf. 13, 413–436.
Altun, M.L., Çitoǧlu, G.S., Yilmaz, B.S., Çoban, T., 2008. Antioxidant properties of
Viburnum opulus and Viburnum lantana growing in Turkey. Int. J. Food Sci. Nutr. 59,
175–180.
Altundag, E., Ozturk, M., 2011. Ethnomedicinal studies on the plant resources of east
Anatolia, Turkey. Procedia - Soc. Behav. Sci. 19, 756–777.
Apak, R., Güçlü, K., Özyürek, M., Karademir, S.E., 2004. Novel total antioxidant capacity
index for dietary polyphenols and vitamins C and E, using their cupric ion reducing
capability in the presence of neocuproine: CUPRAC method. J. Agric. Food Chem. 52,
7970–7981.
Baker, I., Chohan, M., Opara, E.I., 2013. Impact of cooking and digestion, in vitro, on the
antioxidant capacity and anti-inflammatory activity of cinnamon, clove and nutmeg.
Plant Foods Hum. Nutr. 364–369.
Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure
of “antioxidant power”: the FRAP assay. Anal. Biochem. 239, 70–76.
Bhatt, A., Patel, V., 2013. Free Radicals and Antioxidants Antioxidant activity of garlic
using conventional extraction and in vitro gastrointestinal digestion. Free. Radic.
Antioxid. 3, 30–34.
Bouayed, J., Deußer, H., Hoffmann, L., Bohn, T., 2012. Bioaccessible and dialysable
polyphenols in selected apple varieties following in vitro digestion vs. their native
patterns. Food Chem. 131, 1466–1472.
Celep, E., Aydın, A., Yesilada, E., 2012. A comparative study on the in vitro antioxidant
potentials of three edible fruits : cornelian cherry, Japanese persimmon and cherry
laurel. Fodo Chem. Toxicol. 50, 3329–3335.
Celep, E., Aydın, A., Kırmızıbekmez, H., Yesilada, E., 2013. Appraisal of in vitro and in vivo
antioxidant activity potential of cornelian cherry leaves. Food Chem. Toxicol. 62,
448–455.
Celep, E., Charehsaz, M., Akyüz, S., Türköz, E., Yesilada, E., 2015. Effect of in vitro gas-
trointestinal digestion on the bioavailability of phenolic components and the anti-
oxidant potentials of some Turkish fruit wines. Food Res. Int. 78, 209–215.
Celep, E., Akyüz, S., Yesilada, E., 2017. The bioaccessible phenolic profile and antioxidant
potential of Hypericum perfoliatum L. After simulated human digestion Ind. Crops Prod
109, 717–723.
Celep, E., Akyüz, S., Yesilada, E., 2018. Assessment of potential bioavailability of major
phenolic compounds in Lavandula stoechas L. ssp. Stoechas. Ind. Crops Prod. 118,
111–117.
Chen, G., Chen, S., Zhao, Y., Luo, C., Li, J., Gao, Y., 2014. Total phenolic contents of 33
fruits and their antioxidant capacities before and after in vitro digestion. Ind. Crop.
Prod. 57, 150–157.
Eken, A., Yücel, O., Boşgelmez, İ.İ., Baldemir, A., Çubuk, S., Çermik, A.H., Endirli ̇ k,
̇ B.,
Bakir, E., Yildizhan, A., Güler, A., Koşar, M., 2017. Ratlarda akciğer transplanta-
syonda iskemi/reperfüzyonun indüklediği oksidatif hasara karşı Viburnum opulus
meyve ekstresinin koruyucu etkilerinin araştırılması. Kafkas Univ. Vet. Fak. Derg. 23,
437–444.
Erdogan-Orhan, I., Altun, M.L., Sever-Yilmaz, B., Saltan, G., 2011. Anti-acet-
ylcholinesterase and antioxidant assets of the major components (salicin, amento-
flavone, and chlorogenic acid) and the extracts of Viburnum opulus and Viburnum
lantana and their total phenol and flavonoid contents. J. Med. Food 14, 434–440.
Ersoy, N., Ercisli, S., Gundogdu, M., 2017. Evaluation of European Cranberrybush
(Viburnum opulus L.) genotypes for agro-morphological, biochemical and bioactive
characteristics in Turkey. Folia Hortic 29, 181–188.
Farah, A., Monteiro, M., Donangelo, C.M., Lafay, S., 2008. Chlorogenic acids from green
coffee extract are highly bioavailable in humans. J. Nutr. 2, 2309–2315.
Fogliano, V., Verde, V., Randazzo, G., Ritieni, A., 1999. Method for measuring antioxidant
activity and its application to monitoring the antioxidant capacity of wines. J. Agric.
Food Chem. 47, 1035–1040.
Fujita, T., Sezik, E., Tabata, M., Yeşilada, E., Honda, G., Takeda, Y., Tanaka, T., Takaishi,
Y., 1995. Traditional Medicine in Turkey VII: folk medicine in middle and west black
sea Regions. Econ. Bot. 49, 406–422.
Gumienna, M., Lasik, M., Czarnecki, Z., 2011. Bioconversion of grape and chokeberry
wine polyphenols during simulated gastrointestinal in vitro digestion Int. J. Food Sci.
and Nutr. 62, 226–233.
Ilhan, M., Ergene, B., Süntar, I., Özbilgin, S., Çitoğlu, G.S., Demirel, M.A., Keles, H., Altun,
L., Akkol, E., 2014. Preclinical evaluation of antiurolithiatic activity of Viburnum
opulus L. On sodium oxalate-induced urolithiasis rat model. Evid. Based Compl.
Alternat. Med., 578103.
T.H. Barak et al.
Karaçelik, A.A., Küçük, M., Iskefiyeli, Z., Aydemir, S., De Smet, S., Miserez, B., Sandra, P.,
2015. Antioxidant components of Viburnum opulus L. Determined by on-line HPLC-
UV-ABTS radical scavenging and LC-UV-ESI-MS methods. Food Chem. 175, 106–114.
Kraujalis, P., Kraujalienė, V., Kazernavičiūtė, R., Venskutonis, P.R., 2017. Supercritical
carbon dioxide and pressurized liquid extraction of valuable ingredients from
Viburnum opulus pomace and berries and evaluation of product characteristics. J.
Supercrit. Fluids 122, 99–108.
Kraujalyte, V., Venskutonis, P.R., Pukalskas, A., Česoniene, L., Daubaras, R., 2013.
Antioxidant properties and polyphenolic compositions of fruits from different
European cranberrybush (Viburnum opulus L.) genotypes. Food Chem. 141,
3695–3702.
Lachowicz, S., Oszmiański, J., 2018. The influence of addition of cranberrybush juice to
pear juice on chemical composition and antioxidant properties. J. Food Sci. Technol.
55, 3399–3407.
Lafay, S., Gil-izquierdo, A., Manach, C., Morand, C., Besson, C., Scalbert, A., 2006.
Chlorogenic acid is absorbed in its intact form in the stomach of rats. J. Nutr. 136,
1192–1197.
Marrelli, M., Conforti, F., Toniolo, C., Nicoletti, M., Statti, G., Menichini, F., 2014.
Hypericum perforatum: influences of the habitat on chemical composition, photo-in-
duced cytotoxicity, and antiradical activity. Pharm. Biol. 52, 909–918.
Mcdougall, G.J., Fyffe, S., Dobson, P., Stewart, D., 2005. Anthocyanins from red wine –
their stability under simulated gastrointestinal digestion. Phytochemistry 66,
2540–2548.
Oral, R.A., Doǧan, M., Sarioǧlu, K., 2014. Recovery of bioactive phenolic compounds from
olive mill waste water, pomegranate peel, and european cranberrybush (viburnum
opulus L.) juice by preparative MPLC. J. Liq. Chromatogr. Relat. Technol. 37,
1827–1836.
Pavan, V., Aparecida, R., Sancho, S., Pastore, G.M., 2014. The effect of in vitro digestion
on the antioxidant activity of fruit extracts (Carica papaya, Artocarpus heterophillus
and Annona marcgravii). LWT - Food Sci. Tech. 59, 1247–1251.
69
Industrial Crops & Products 131 (2019) 62–69
Perova, I.B., Zhogova, A.A., Cherkashin, A.V., Éller, K.I., Ramenskaya, G.V., Samylina,
I.A., 2014. Biologically active substances from European Guelder berry fruits. Pharm.
Chem. J. 48, 332–339.
Prieto, P., Pineda, M., Aguilar, M., 1999. Spectrophotometric quantitation of antioxidant
capacity through the formation of a phosphomolybdinum complex: specific appli-
cation to the determination of Vitamin E. Anal. Biochem. 269, 337–341.
Rice-Evans, C., Miller, N.J., Paganga, G., 1997. Antioxidant properties of phenolic com-
pounds. Trends Plant Sci. 2, 152–159.
Rop, O., Reznicek, V., Valsikova, M., Jurikova, T., Mlcek, J., Kramarova, D., 2010.
Antioxidant properties of European cranberrybush fruit (Viburnum opulus var. edule).
Molecules 15, 4467–4477.
Sagdic, O., Aksoy, A., Ozkan, G., 2006. Evaluation of the antibacterial and antioxidant
potentials of cranberry (gilaburu, Viburnum opulus L.) fruit extract. Acta Aliment. 35,
487–492.
Sarıözkan, S., Türk, G., Eken, A., Bayram, L.Ç., Baldemir, A., Doğan, G., 2017. Gilaburu
(Viburnum opulus L.) fruit extract alleviates testis and sperm damages induced by
taxane-based chemotherapeutics. Biomed. Pharmacother. 95, 1284–1294.
Tanaka, T., Higa, S., Hirano, T., Kotani, M., Matsumoto, M., Fujita, A., Kawase, I., 2003.
Flavonoids as potential anti-allergic substances. Cur. Med. Chem. 2, 57–69.
Ulger, H., Ertekin, T., Karaca, O., Canoz, O., Nisari, M., 2012. Influence of gilaburu
(Viburnum opulus) juice on 1,2-dimethylhydrazine (DMH)-induced colon cancer. Tox.
Ind. Health 29, 824–829.
Vallejo, F., Gil-Izquierdo, A., Perez-Vicente, A., Garcia-Viguera, C., 2004. In vitro gas-
trointestinal digestion study of broccoli inflorescence phenolic compounds, glucosi-
nolates, and Vitamin C. J. Agric. Food Chem. 52, 135–138.
Velioglu, Y.S., Ekici, L., Poyrazoglu, E.S., 2006. Phenolic composition of European
cranberrybush (Viburnum opulus L.) berries and astringency removal of its commer-
cial juice. Int. J. Food Sci. Technol. 41, 1011–1015.
Wilcox, C.S., 2005. Oxidative stress and nitric oxide deficiency in the kidney: a critical
link to hypertension? Am. J. Physiol. Regul. Integr. Comp. Physiol. 289, 913–935.