Hindawi
Oxidative Medicine and Cellular Longevity
Volume 2018, Article ID 3482521, 16 pages
https://doi.org/10.1155/2018/3482521
Research Article
Multifunctional Phytocompounds in Cotoneaster Fruits:
Phytochemical Profiling, Cellular Safety, Anti-Inflammatory and
Antioxidant Effects in Chemical and Human Plasma
Models In Vitro
Agnieszka Kicel ,1 Joanna Kolodziejczyk-Czepas,2 Aleksandra Owczarek,1
Magdalena Rutkowska,1 Anna Wajs-Bonikowska,3 Sebastian Granica,4 Pawel Nowak,2
and Monika A. Olszewska 1
1
Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Lodz, 1 Muszynskiego, 90-151 Lodz, Poland
Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143,
90-236 Lodz, Poland
3
Institute of General Food Chemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, 4/
10 Stefanowskiego, 90-924 Lodz, Poland
4
Department of Pharmacognosy and Molecular Basis of Phytotherapy, Faculty of Pharmacy, Medical University of Warsaw,
1 Banacha, 02-097 Warsaw, Poland
2
Correspondence should be addressed to Agnieszka Kicel; agnieszka.kicel@umed.lodz.pl
Received 22 June 2018; Accepted 30 August 2018; Published 24 October 2018
Academic Editor: Felipe L. de Oliveira
Copyright © 2018 Agnieszka Kicel et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The work presents the results of an investigation into the molecular background of the activity of Cotoneaster fruits, providing a
detailed description of their phytochemical composition and some of the mechanisms of their anti-inflammatory and antioxidant
effects. GS-FID-MS and UHPLC-PDA-ESI-MS3 methods were applied to identify the potentially health-beneficial constituents of
lipophilic and hydrophilic fractions, leading to the identification of fourteen unsaturated fatty acids (with dominant linoleic acid,
375.4–1690.2 mg/100 g dw), three phytosterols (with dominant β-sitosterol, 132.2–463.3 mg/100 g), two triterpenoid acids
(10.9–54.5 mg/100 g), and twenty-six polyphenols (26.0–43.5 mg GAE/g dw). The most promising polyphenolic fractions
exhibited dose-dependent anti-inflammatory activity in in vitro tests of lipoxygenase (IC50 in the range of 7.7–24.9 μg/U) and
hyaluronidase (IC50 in the range of 16.4–29.3 μg/U) inhibition. They were also demonstrated to be a source of effective
antioxidants, both in in vitro chemical tests (DPPH, FRAP, and TBARS) and in a biological model, in which at in vivo-relevant
levels (1–5 μg/mL) they normalized/enhanced the nonenzymatic antioxidant capacity of human plasma and efficiently protected
protein and lipid components of plasma against peroxynitrite-induced oxidative/nitrative damage. Moreover, the investigated
extracts did not exhibit cytotoxicity towards human PMBCs. Among the nine Cotoneaster species tested, C. hjelmqvistii, C.
zabelii, C. splendens, and C. bullatus possess the highest bioactive potential and might be recommended as dietary and
functional food products.
1. Introduction
Edible fruits are widely recognized as a valuable source of
structurally diverse phytochemicals with a broad spectrum
of health-promoting properties. Decreased cholesterol levels,
lower blood pressure, better mental health, and protection
against cancer are only a few of the many benefits associated
with the regular intake of fruit products, as indicated by
numerous epidemiological studies [1]. Among the different
fruit-bearing families, the Rosaceae seems to be of special
importance. With over 3000 species, the family provides
numerous types and varieties of fruits, some of which, such
2
Oxidative Medicine and Cellular Longevity
(a)
(b)
Figure 1: The fruits of C. bullatus (a) and C. splendens (b).
as apples, pears, strawberries and cherries, have great
economic and dietary importance, and are frequently and
willingly consumed due to their excellent flavors and proven
nutritional value [2]. Many other taxa (e.g., Aronia sp.,
Sorbus sp., Pyracantha sp., and Prunus spinosa L.) produce
fruits, that while less attractive in taste and appearance, are,
nonetheless, distinguished by especially high quantities of
bioactive constituents, which makes them perfect candidates
for more specialized food applications, for example, as functional food products or food additives [3–6].
The chemical diversity of health-beneficial phytochemicals contained in rosaceous plant materials is immense and
ranges from highly lipophilic to strongly polar constituents.
Unsaturated fatty acids of almond oil, the cholesterolregulating phytosterols of Prunus africana (Hook.f.) Kalkman,
and the pentacyclic triterpenes, ubiquitous throughout the
Rosaceae, with proven anti-inflammatory activity are some
examples of the possible structures from the hydrophobic
end of the spectrum [7, 8]. On the other hand, the hydrophilic
fractions often contain an abundance of highly-valued polyphenol antioxidants belonging to numerous chemical classes,
such as flavonoids, phenolic acids, and tannins. The bioactive
potential of Rosaceae fruits is, therefore, associated not with a
single fraction but rather is an effect of the presence of a range
of phytochemicals.
The genus Cotoneaster Medikus is one of the largest
genera of the Rosaceae family (subfamily Spiraeoideae, tribe
Pyreae) comprising about 500 species of shrubs or small
trees. Its members are native to the Palearctic region
(temperate Asia, Europe, north Africa) but are often cultivated throughout Europe as ornamental plants due to their
decorative bright red fruits (Figure 1). The center of diversity
of the taxon are the mountains of southwestern China and
the Himalayas [9, 10], where the fruits have been used for
culinary purposes by the local communities. The nutritional
value of the fruits as a source of vitamins and minerals has
been confirmed [11, 12] and additional beneficial health
effects of the fruit consumption have been also reported in
the traditional medicine for the treatment of diabetes mellitus, cardiovascular diseases, nasal hemorrhage, excessive
menstruation, fever, and cough [9, 10]. The phytochemical
research on the subject is scarce, but the available data indicate the tendency of the fruits to accumulate a wide range
of active metabolites. In particular, the fruits of Cotoneaster
pannosus Franch. are a source of linoleic acid, those of
Cotoneaster microphylla Wall ex Lindl contain pentacyclic
triterpenoids, and the polyphenolic fractions of C. pannosus
and Cotoneaster integerrimus Medik. fruits are rich in epicatechin, shikimic acid, and chlorogenic acid [9, 11, 12].
However, broader generalization of their properties is troublesome, and the possible wider application of the fruits, for
example, as functional food products, is hindered by a lack
of systematic studies. Similarly limited is the information
on the activity of Cotoneaster fruits. Preliminary studies
have been performed on the fruits of C. integerrimus and
C. pannosus with regard to their antioxidant, anticholinesterase, antityrosinase, antiamylase, and antiglucosidase properties, and their free radical-scavenging potential was proven to
be the most promising [9, 12]. Still, the research was carried
out using only simple in vitro chemical tests and did not
cover in vivo-relevant antioxidant mechanisms.
The aim of this study was, therefore, to provide a more
detailed insight into the chemical composition and activity
of Cotoneaster fruits. To this end, the fruits from nine species
of Cotoneaster cultivated in Poland were analyzed for a range
of lipophilic and hydrophilic (polyphenolic) constituents
with acknowledged health-promoting properties using a
combination of chromatographic and spectroscopic methods
(GC-FID-MS, UHPLC-PDA-ESI-MS3, and UV-Vis spectrophotometry). The most promising polyphenolic fractions
were then subjected to an analysis of antioxidant activity
comprising eight complementary in vitro tests (both chemical
and biological plasma models) covering some of the mechanisms crucial for reducing the level of oxidative damage in
the human organism, that is, scavenging of free radicals,
enhancement of the nonenzymatic antioxidant capacity of
blood plasma, and protection of its lipid and protein components against oxidative/nitrative changes. Additionally, the
inhibitory effects of the fruit extracts on the proinflammatory
enzymes, that is, lipoxygenase and hyaluronidase, were also
measured. Finally, the cellular safety of the extracts was evaluated in cytotoxicity tests employing human peripheral blood
mononuclear cells (PMBCs).
2. Materials and Methods
2.1. Plant Material. The fruit samples of nine selected
Cotoneaster Medik. species, that is, C. lucidus Schltdl. (AR),
C. divaricatus Rehder et E.H. Wilson (BG), C. horizontalis
Decne. (BG), C. nanshan Mottet (BG), C. hjelmqvistii Flinck
Oxidative Medicine and Cellular Longevity
et B. Hylmö (BG), C. dielsianus E. Pritz. (BG), C. splendens
Flinck et B. Hylmö (BG), C. bullatus Bois (BG), and C. zabelii
C.K. Schneid. (BG) were collected in September 2013, in the
Botanical Garden (BG; 51°45′N 19°24′E) in Lodz (Poland)
and in the Arboretum (AR; 51°49′N 19°53′E), Forestry
Experimental Station of Warsaw University of Life Sciences
(SGGW) in Rogow (Poland). The voucher specimens were
deposited in the Herbarium of the Department of Pharmacognosy, Medical University of Lodz (Poland). The raw
materials were powdered with an electric grinder, sieved
through a 0.315 mm sieve, and stored in airtight containers
until use.
2.2. General. Reagents and standards of analytical or
HPLC grade such as 2,2-diphenyl-1-picrylhydrazyl (DPPH),
2,4,6-tris-(2-pyridyl)-s-triazine (TPTZ), 2,2′-azobis-(2amidinopropane)-dihydrochloride (AAPH), linoleic acid,
2-thiobarbituric acid, Tween® 40, 5,5′-dithiobis-(2-nitrobenzoic acid) (DNTB), xylenol orange disodium salt,
Histopaque®-1077 medium N,O-bis-(trimethylsilyl)-trifluoroacetamide with 1% 1-trimethylchlorosilane (BSTFA +
TMCS), boron trifluoride, bovine testis hyaluronidase, lipoxygenase from soybean, reference standards of fatty acid
methyl esters (FAMEs), ethyl oleate, 5-α-cholesterol, (±)-6hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
acid
(Trolox®), butylated hydroxyanisole (BHA), 2,6-di-tertbutyl-4-methylphenol (BHT), gallic acid monohydrate,
quercetin dehydrate, chlorogenic acid hemihydrate (5-Ocaffeoylquinic acid), 3-O- and 4-O-caffeoylquinic acids,
hyperoside semihydrate, isoquercitrin, rutin trihydrate, procyanidins B-2 and C-1, (−)-epicatechin, and indomethacin
were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The standards of quercetin 3-O-β-D-(2″-O-β-D-xylosyl)galactoside and quercitrin (quercetin 3-O-α-L-rhamnoside)
have previously been isolated in our laboratory from C.
bullatus and C. zabelii leaves with at least 95% HPLC purity
(unpublished results). A (Ca2+ and Mg2+)-free phosphate
buffered saline (PBS) was purchased from Biomed (Lublin,
Poland). Peroxynitrite was synthesized according to Pryor
et al. [13]. Anti-3-nitrotyrosine polyclonal antibody, biotinconjugated secondary antibody, and streptavidin/HRP were
purchased from Abcam (Cambridge, UK). HPLC grade solvents such as acetonitrile and formic acid were from Avantor
Performance Materials (Gliwice, Poland). For chemical tests,
the samples were incubated at a constant temperature using a
BD 23 incubator (BINDER, Tuttlingen, Germany) and measured using a UV-1601 Rayleigh spectrophotometer (Beijing,
China). Activity tests in blood plasma models and enzyme
inhibitory assays were performed using 96-well plates and
monitored using a SPECTROStar Nano microplate reader
(BMG LABTECH, Ortenberg, Germany).
2.3. Phytochemical Profiling
2.3.1. Extraction and Derivatization of Lipophilic
Phytochemicals. The fruit samples (7.0 g) were exhaustively
extracted in a Soxhlet apparatus with chloroform (150 mL,
24 h), to give lipid extracts (288–467 mg dw), which were
then subjected to quantification of lipophilic compounds.
3
Fatty acids were assayed as fatty acid methyl esters (FAMEs)
prepared according to a method described earlier [14].
Phytosterols and triterpenes were assayed after their transformation to trimethylsilyl ethers (TMSs) according to Thanh
et al. [15]. The FAME and TMS mixtures were independently
analyzed by GC-FID-MS.
2.3.2. GC-FID-MS Analysis. The analyses of lipophilic fractions were performed on a Trace GC Ultra instrument
coupled with a DSQII mass spectrometer (Thermo Electron,
Waltham, MA, USA) and a MS-FID splitter (SGE Analytical
Science, Trajan Scientific Americas, Austin, TX, USA). The
applied mass range was 33–550 amu, ion source-heating
was 200°C, and ionization energy was 70 eV. The conditions
for FAMEs were as follows: capillary column: TG-WaxMS
(30 m × 0.25 mm i.d., film thickness 0.25 μm; Thermo Fisher
Scientific, Waltham, MA, USA); temperature program:
3–30 min: 50–240°C at 4°C/min; and injector and detector
temperatures: 250°C and 260°C, respectively. The conditions for TMSs were as follows: capillary column: HP-5
(30 m × 0.25 mm i.d., film thickness 0.25 μm; Agilent Technologies, Santa Clara, CA, USA); temperature program:
1–15 min: 100–250°C, at 10°C/min; 15–30 min: 250–300°C,
at 4°C/min; and injector and detector temperatures: 310°C
and 300°C, respectively. In all cases, the carrier gas was
helium (constant pressure: 300 kPa). The lipophilic analytes
were identified by comparison of their MS profiles with those
stored in the libraries NIST 2012 and Wiley Registry of Mass
Spectral Data (10th and 11th eds). Retention times (t R ) of
FAMEs were also compared with those of the commercial
FAME mixture. The analyte levels were expressed as
mg/100 g fruit dry weight (dw), calculated using the internal
standards of ethyl oleate and 5-α-cholesterol (for the fatty
acids as well as phytosterols and triterpenoids, respectively)
and it was recalculated to the content in the plant material
taking into account the extraction yield.
2.3.3. Extraction of Polyphenolic Compounds. The fruit samples (100–500 mg) were first defatted by preextraction with
chloroform (20 mL, 15 min; the chloroform extracts were discarded), then refluxed for 30 min with 30 mL of 70% (v/v)
aqueous methanol, and twice for 15 min with 20 mL of the
same solvent. The combined extracts were diluted with the
extractant to 100 mL. Each sample was extracted in triplicate
to give the test extracts, which were analyzed for their total
phenolic contents (TPCs) and antioxidant activity in chemical models. For UHPLC analyses and antioxidant activity
evaluation in the human plasma models, the test extracts
were evaporated in vacuo and lyophilized using an Alpha
1-2/LDplus freeze dryer (Christ, Osterode am Harz, Germany)
before weighing.
2.3.4. UHPLC-PDA-ESI-MS3 Analysis. Metabolite profiling
was performed on an UltiMate 3000 RS UHPLC system
(Dionex, Dreieich, Germany) with PDA detector scanning
in the wavelength range of 220–450 nm and an amaZon
SL ion trap mass spectrometer with ESI interface (Bruker
Daltonics, Bremen, Germany). Separations were carried
out on a Kinetex XB-C18 column (150 × 2.1 mm, 1.7 μm;
4
Phenomenex Inc., Torrance, CA, USA). The mobile phase
consisted of solvent A (water-formic acid, 100 : 0.1, v/v)
and solvent B (acetonitrile-formic acid, 100 : 0.1, v/v) with
the following elution profile: 0–45 min, 6–26% (v/v) B;
45–55 min, 26–95% B; 55–60 min, 95% B; and 60–63 min,
95–6% B. The flow rate was 0.3 mL/min. The column
temperature was 25°C. Before injections, samples of dry
extracts (15 mg) were dissolved in 1.5 mL of 70% aqueous
methanol, filtered through PTFE syringe filters (25 mm,
0.2 μm, Vitrum, Czech Republic) and injected (3 μL) into
the UHPLC system. UV-Vis spectra were recorded over
a range of 200–600 nm, and chromatograms were acquired
at 280, 325, and 350 nm. The LC eluate was introduced
directly into the ESI interface without splitting and analyzed
in a negative ion mode using a scan from m/z 70 to 2200. The
MS2 and MS3 fragmentations were obtained in Auto MS/MS
mode for the most abundant ions at the time. The nebulizer
pressure was 40 psi, dry gas flow was 9 L/min, dry temperature was 300°C, and capillary voltage was 4.5 kV.
2.3.5. Determination of Total Phenolic Content (TPC). The
TPC levels were determined according to the FolinCiocalteu method as described previously [16]. The results
were expressed as mg of gallic acid equivalents (GAE) per g
of dry weight of the plant material (mg GAE/g dw).
2.4. Lipoxygenase (LOX) and Hyaluronidase (HYAL)
Inhibition Tests. The ability of the fruit extracts to inhibit
lipoxygenase (LOX) and hyaluronidase (HYAL) was evaluated according to the method optimized earlier [17]. The
results of both tests were expressed as IC50 values (μg/mL)
from concentration-inhibition curves.
2.5. Antioxidant Activity in Chemical Models. The DPPH
free-radical scavenging activity was determined according
to a previously optimized method [16] and expressed as
normalized EC50 values calculated from concentrationinhibition curves. The FRAP (ferric reducing antioxidant
power) was determined according to [16] and expressed in
μmol of ferrous ions (Fe2+) produced by 1 g of the dry extract
or standard, which was calculated from the calibration curve
of ferrous sulfate. The ability of the extracts to inhibit AAPHinduced peroxidation of linoleic acid was assayed as
described previously [18] with peroxidation monitored by
quantification of thiobarbituric acid-reactive substances
(TBARS) according to a previously optimized method [19],
and the antioxidant activity was expressed as IC50 values calculated from concentration-inhibition curves. Additionally,
the activity parameters in all of the assays were also expressed
as μmol Trolox® equivalents (TE) per g of dry weight of the
plant material (μmol TE/g dw).
2.6. Antioxidant Activity in Human Plasma Models
2.6.1. Isolation of Blood Plasma and Sample Preparation.
Blood (buffy coat units) from eight healthy volunteers,
received from the Regional Centre of Blood Donation and
Blood Treatment in Lodz (Poland), was centrifuged to obtain
plasma [20]. All experiments were approved by the committee on the Ethics of Research at the Medical University of
Oxidative Medicine and Cellular Longevity
Lodz RNN/347/17/KE. Plasma samples, diluted with 0.01 M
Tris/HCl pH 7.4 (1 : 4 v/v), were preincubated for 15 min at
37°C with the examined extracts, added to the final concentration range of 1–50 μg/mL, and then exposed to 100 or
150 μM peroxynitrite (ONOO−). Control samples were prepared with plasma untreated with the extracts and/or peroxynitrite. To eliminate the possibility of direct interactions of
the extracts with plasma proteins and lipids, several experiments with blood plasma and the extracts only (without
adding ONOO−) were also performed and no prooxidative
effect was found.
2.6.2. Determination of 3-Nitrotyrosine and Thiols in
Human Plasma Proteins. The peroxynitrite-induced protein
damage in blood plasma was determined by the use of
3-nitrotyrosine and protein thiol levels (−SH) as biomarkers
of oxidative stress. Immunodetection of 3-nitrotyrosinecontaining proteins by the competitive ELISA (C-ELISA)
method in plasma samples (control or antioxidants and
100 μM ONOO−-treated plasma) was performed according
to [20]. The nitrofibrinogen (3NT-Fg, at a concentration of
0.5 μg/mL and 3–6 mol nitrotyrosine/mol protein) was prepared for use in the standard curve. The concentrations of
nitrated proteins that inhibit antinitrotyrosine antibody
binding were estimated from the standard curve and are
expressed as the 3NT-Fg equivalents (in nmol/mg of
plasma protein). The concentration of free thiol groups
(−SH) in plasma samples (control or antioxidants and
100 μM ONOO−-treated plasma) was measured spectrophotometrically according to Ellman’s method [20]. The free
thiol group concentration was calculated from the standard
curve of glutathione (GSH) and expressed as umol/mL of
plasma.
2.6.3. Determination of Lipid Hydroperoxides and TBARS in
Human Blood Plasma. The peroxynitrite-induced lipid peroxidation in blood plasma was determined spectrophotometrically by evaluation of the level of lipid hydroperoxides and
TBARS. The concentration of hydroperoxides in plasma
samples (control or antioxidants and 100 μM ONOO−treated plasma) was determined by a ferric-xylenol orange
(FOX-1) protocol with a later modification [20]. The amount
of lipid hydroperoxides was calculated from the standard
curve of hydrogen peroxide and expressed in nmol/mg of
plasma proteins. Determination of TBARS in plasma samples (control or antioxidants and 100 μM ONOO−-treated
plasma) was performed according to [20]. The TBARS values
were expressed in μmol TBARS/mL of plasma.
2.6.4. Ferric Reducing Ability of Human Blood Plasma
(FRAP). The influence of the extracts on the nonenzymatic
antioxidant status of plasma was conducted by measurements of their ability to reduce ferric ions (Fe3+) to ferrous
ions (Fe2+). The experiments were performed according to
Benzie and Strain [21] and modified by KolodziejczykCzepas et al. [20]. The FRAP values of plasma samples
(control or antioxidants and 150 μM ONOO−-treated
plasma) were expressed in mM Fe2+ in plasma as calculated
from the calibration curve of ferrous sulphate.
Oxidative Medicine and Cellular Longevity
5
3. Results and Discussion
are in accordance with previous reports for the fruits of
C. pannosus from Italy, as well as the branches of C. horizontalis Decke. of Egyptian origin and the seeds of C. bullatus,
C. dielsianus, C. francheti Bois, C. moupinensis Franch., and
C. simonsii Baker cultivated in Germany, in which linoleic
and palmitic acids were also detected as the major fatty acid
components [9, 22, 23].
The unsaturated fatty acids are known factors associated
with the prevention of various chronic and acute diseases,
such as cardiovascular diseases, osteoporosis, immune disorders, and cancer [7]. Linoleic acid, the representative of the
omega-6 fatty acid family (essential fatty acids (EFA)), is considered a vital constituent of a healthy human diet, due to its
contribution to cholesterol metabolism (regulation of plasma
total cholesterol and low-density lipoprotein cholesterol
levels and HDL-LDL ratio) and its association with a lower
risk of atherosclerosis [24]. Main sources of this compound
are plant oils, derived, inter alia, from the seeds of safflower,
sunflower, grape, pumpkin, and corn. The available literature
data [25, 26] indicate that whole fruits of some Rosaceae members, such as Crataegus monogyna Jacq., Prunus spinosa L.,
and Rubus ulmifolius Schott., might be considered as abundant in linoleic acid, constituting over 10% of their lipophilic
fraction [26]. Our present results indicate that the analyzed
Cotoneaster fruits also deserve more attention as rich sources
of this compound.
3.1. GC-FID-MS Analysis of Fatty Acids. The fatty acid
profiles of the lipophilic fractions in the chloroform extracts
of the Cotoneaster fruits were determined by GC-FID-MS
analysis of methyl ester derivatives (FAMEs). As shown in
Table 1 and Figure 2, fourteen fatty acids were identified,
including saturated, mono-, and polyunsaturated acids with
chain lengths ranging from 6 to 22 carbon atoms. Their total
content (TFA) varied among the Cotoneaster species from
902.5 to 2683.8 mg/100 g of fruit dry weight (dw) with the
highest levels noted for C. zabelii (2683.8 mg/100 g dw) and
C. splendens (2024.1 mg/100 g dw). All analyzed fruits contained primarily poly- and monounsaturated acids, constituting 41.6–66.8% and 18.6–29.6% of TFA, respectively.
The major component in each sample was linoleic acid
C18 : 2 Δ9,12, the sole representative of the polyunsaturated
acids. Its content varied among species from 375.4 to
1690.2 mg/100 g fruit dw with the highest amounts (above
10 mg/g dw) recorded for the fruits of C. zabelii, C. splendens,
C. hjelmqvistii, and C. horizontalis. Relatively high levels of
oleic acid C18 : 1 Δ9, a monounsaturated acid, were also
noted, especially for the C. zabelii and C. splendens (649.7
and 473.7 mg/100 g dw, respectively). Regarding saturated
acids, they accounted for only 12.3–28.8% of TFA. The
highest content of this group was observed in the fruits
of C. zabelii, C. splendens, and C. nanshan, with palmitic
acid C16 : 0 being the dominant compound (226.5, 212.6
and 168.7 mg/100 g dw, respectively).
The present work is the first comparison of several
Cotoneaster fruits in terms of their fatty acid profile.
Despite some quantitative differences observed between the
investigated fruits, a high level of consistency can be noticed
in the qualitative composition of this fraction. The results
3.2. GC-FID-MS Analysis of Phytosterols and Triterpenoids.
Apart from fatty acids, three phytosterols (campesterol,
β-sitosterol, and stigmasterol) and four triterpenes (α- and
β-amyrins, ursolic and oleanolic acids) were identified in
the chloroform extracts of the Cotoneaster fruits, based on
GC-FID-MS analysis of their trimethylsilyl ether derivatives
(TMSs). As reported in Table 2 and Figure 2, the total
content of sterols and triterpenoids, depending on the tested
species, was in the range of 154.6–515.6 mg/100 g of fruit
(dw) with the highest levels observed for C. splendens
(515.6 mg/100 g dw) and C. nanshan (438.0 mg/100 g dw).
The dominant compound in all samples was β-sitosterol,
with the levels ranging from 132.2 to 463.3 mg/100 g dw
(76.5–89.3% of the total sterols and triterpenes). The highest
content of β-sitosterol was observed for the fruit of C. splendens (463.3 mg/100 g dw) followed by those of C. nanshan
(391.3 mg/100 g dw) and C. horizontalis (316.3 mg/100 g dw).
Other individual components were observed at much lower
concentrations, reaching at most 42 mg/100 g dw.
Regarding the phytosterol and triterpenoid profile, the
present results are generally similar to the data obtained
previously for different organs of Cotoneaster species,
although some differences can be noticed in relative proportions of particular compounds. Among the sterols and
triterpenoids identified earlier for the C. horizontalis
branches collected in Egypt, α-amyrin was the dominant
compound, constituting 14.4% of the total lipophilic constituents, followed by β-sitosterol (8.5%) and stigmasterol
(1.1%) [23]. The ursolic acid was isolated previously from
C. simonsii twigs [27], C. racemiflora Desf. twigs [28], and
C. microphylla fruits [11], but the present work is the first
to describe its quantitative levels in the Cotoneaster plants.
2.7. Cellular Safety Testing. The cytotoxicity of the examined
extracts was conducted in an experimental system of peripheral blood mononuclear cells (PBMCs). PBMCs were isolated
from fresh human blood using the Histopaque®-1077
medium, according to a procedure described in our previous
work [19]. Then, the cells (1 × 106 PBMCs/mL, suspended in
PBS) were incubated with Cotoneaster fruit extracts at the
final concentrations of 5, 25, and 50 μg/mL. Measurements
of cell viability were executed after two, four, and six hours
of incubation (at 37°C) in a routine dye excluding test, based
on a staining with 0.4% Trypan blue. The procedure was carried out according to the manufacturer’s protocol using a
microchip-type automatic cell counter Bio-Rad (Hercules,
CA, USA).
2.8. Statistical and Data Analysis. The statistical analysis was
performed using STATISTICA 13Pl software for Windows
(StatSoft Inc., Krakow, Poland). The results were reported
as means ± standard deviation (SD) or ±standard error (SE)
for the indicated number of experiments. The significance
of differences between the samples and controls were analyzed by one-way ANOVA, followed by the post hoc Tukey’s
test for multiple comparison. A level of p < 0 05 was accepted
as statistically significant.
6
Table 1: Content of fatty acids (mg/100 g dw) in the Cotoneaster fruits.a
Fruit sample
C. lucidus
C. divaricatus
8:0
12 : 0
14 : 0
3.41
± 0.10B
2.24
± 0.11A
2.35
± 0.10F
0.61
± 0.03A,B
0.69
± 0.01B
0.65
± 0.01B
1.12
± 0.04C
1.77
± 0.03E
1.55
± 0.04D
0.51
± 0.01A
1.44
± 0.05D
2.77
± 0.01F
0.82
± 0.05A
2.28
± 0.10E
1.73
± 0.10C,D
1.96
± 0.10D
1.58
± 0.08C
2.80
± 0.15F
1.53
± 0.04C
1.08
± 0.05B
5.97
± 0.30E
2.86
± 0.15A
4.80
± 0.25D
4.33
± 0.20C,D
4.48
± 0.32D
4.14
± 0.25B,C,D
5.59
± 0.32E
3.73
± 0.22B,C
3.61
± 0.15B
C. horizontalis
tr
C. nanshan
nd
C. hjelmqvistii
tr
C. dielsianus
6.11
± 0.20C
C. splendens
tr
C. bullatus
tr
C. zabelii
3.25
± 0.11B
15 : 0
nd
tr
tr
0.65
± 0.01A
nd
0.59
± 0.02A
nd
0.51
± 0.03A
nd
16 : 0
126.25
± 5.23A
136.65
± 6.20A
174.10
± 5.40B
168.68
± 6.40B
174.55
± 8.00B
177.81
± 6.43B
212.60
± 11.00C
120.49
± 5.20A
226.45
± 5.40C
17 : 0
tr
0.82
± 0.01B
1.37
± 0.05C
2.16
± 0.12D
0.56
± 0.03A
3.15
± 0.16E
2.18
± 0.11D
2.37
± 0.10D
1.44
± 0.06C
16 : 1 Δ9
18 : 0
18 : 1 Δ9
20 : 0
18 : 2 Δ9,12
8.96
± 0.51E
4.90
± 0.21C
8.68
± 0.43E
8.22
± 0.38D,E
3.36
± 0.16A,B
4.14
± 0.19B,C
8.39
± 0.50D,E
2.54
± 0.12A
7.58
± 0.35D
92.98
± 5.12F
63.23
± 2.45E
38.38
± 2.10B,C
87.58
± 3.54F
41.19
± 2.05C
37.85
± 1.04B,C
32.95
± 1.14A,B
29.83
± 1.10A
53.09
± 2.70D
258.05
± 12.11A,B
262.09
± 10.09B
294.05
± 13.01B,C
384.72
± 18.12D
335.10
± 14.10C
273.21
± 15.02B
473.70
± 20.01E
215.22
± 10.00A
649.73
± 25.05F
6.02
± 0.20A
6.73
± 0.31A
13.25
± 0.60C
17.30
± 0.75D
17.09
± 0.71D
16.76
± 0.80D
18.34
± 0.61D
10.85
± 0.55B
30.34
± 1.32E
375.35
± 18.01A
566.60
± 25.03B
1012.83
± 45.02D
736.79
± 30.01C
1216.27
± 50.01E
643.22
± 15.15B,C
1225.89
± 30.12E
677.53
± 16.15C
1690.23
± 55.01F
20 : 1 Δ11
tr
2.04
± 0.08B
tr
3.89
± 0.20D
0.56
± 0.02A
0.79
± 0.03A
2.49
± 0.11C
4.07
± 0.15D
5.78
± 0.21E
22 : 0
18.77
± 0.80B
7.55
± 0.22A
26.05
± 1.00C
19.25
± 0.95B
24.38
± 1.05C
19.91
± 0.55B
37.30
± 1.85E
30.33
± 1.10D
9.75
± 0.20A
a
Values presented as means ± SD calculated per dw of the plant material (n = 3); tr—trace, the content less than 0.5 mg/100 g dw; nd—not detected; different capital letters within the same row indicate significant
differences at α = 0 05 in HSD Tukey’s test; 6 : 0—caproic acid, 8 : 0—caprylic acid, 12 : 0—lauric acid, 14 : 0—myristic acid, 15 : 0—pentadecylic acid, 16 : 0—palmitic acid, 17 : 0—margaric acid, 16 : 1
Δ9—palmitoleic acid, 18 : 0—stearic acid, 18 : 1 Δ9—oleic acid, 20 : 0—arachidic acid, 18 : 2 Δ9,12—linoleic acid, 20 : 1 Δ11—eicosenoic acid and 22 : 0—behenic acid.
Oxidative Medicine and Cellular Longevity
6:0
7
50
FA, PST + TR, TPC (mg/g dw)
43.5
40
30
26.8
26.0
20
10
5.2
9.0
0
1.5
FA
PST + TR
DPPH (EC50 휇g/mL), FRAP (휇mol Fe2+/g dw),
TBARS (IC50, 휇g/mL), TE (휇mol TX/g dw)
Oxidative Medicine and Cellular Longevity
TPC
1200
1089.8
1000
800
600
543.9
608.2
434.2
400
241.4
200
0
178.4
165.8
213.4
205.3
85.1
62.9
62.5
DPPH TE (DPPH) FRAP TE (FRAP) TBARS TE (TBARS)
Median
First quartile (Q1)
Min-max
Median
First quartile (Q1)
Min-max
(a)
(b)
Figure 2: Variability of the measured quantitative and activity parameters among the investigated Cotoneaster fruits. (a) FA, total fatty acids;
PS + TR, sum of phytosterols and tritrepenes; TPC, total phenolic content, expressed in gallic acid equivalents (GAE). (b) DPPH, radical
scavenging activity expressed as EC50 value; FRAP, ferric reducing antioxidant power; TBARS, inhibition of linoleic acid peroxidation;
TE, Trolox® equivalent antioxidant activity.
Table 2: Content of phytosterols and triterpenes (mg/100 g dw) in the Cotoneaster fruits.a
Fruit sample
C. lucidus
C. divaricatus
C. horizontalis
C. nanshan
C. hjelmqvistii
C. dielsianus
C. splendens
C. bullatus
C. zabelii
Campesterol
C
6.83 ± 0.30
9.06 ± 0.31E
6.04 ± 0.22B,C
8.94 ± 0.40E
4.31 ± 0.12A
5.38 ± 0.21B
13.11 ± 0.56F
7.98 ± 0.31D
6.77 ± 0.30C
Stigmasterol
β-Sitosterol
B
195.31 ± 5.31
132.19 ± 4.23A
316.31 ± 15.03D
391.26 ± 17.02E
211.99 ± 10.13B
181.96 ± 5.22B
463.26 ± 15.10F
274.47 ± 12.15C
273.25 ± 10.22C
nd
nd
nd
nd
nd
tr
nd
2.70 ± 0.07B
1.00 ± 0.01A
β-Amyrin
nd
nd
nd
nd
1.17 ± 0.05A
2.12 ± 0.10B
nd
0.88 ± 0.04A
nd
α-Amyrin
A
1.05 ± 0.05
2.48 ± 0.07B
0.88 ± 0.02A
5.26 ± 0.21C
14.37 ± 0.61F
6.32 ± 0.24D
8.79 ± 0.30E
14.15 ± 0.50F
14.89 ± 0.22F
Ursolic acid
Oleanolic acid
B
15.52 ± 0.53D
8.65 ± 0.32A,B
17.24 ± 0.50E
26.52 ± 1.05F
18.41 ± 0.50E
7.30 ± 0.18A
17.05 ± 0.45D,E
13.05 ± 0.52C
9.27 ± 0.36B
6.61 ± 0.30
2.21 ± 0.04A
25.45 ± 1.10F
6.04 ± 0.22B
27.03 ± 0.98F
10.49 ± 0.35C
13.42 ± 0.45D
41.45 ± 1.50G
20.70 ± 1.03E
a
Values presented as means ± SD calculated per dw of the plant material (n = 3); tr—trace, the content less than 0.5 mg/100 g dw; nd—not detected; different
capital letters within the same row indicate significant differences at α = 0 05 in HSD Tukey’s test.
On the other hand, betulinic acid, reported earlier for
C. microphylla fruits [11], was not detected during the present study in any fruit sample.
Phytosterols (β-sitosterol, stigmasterol, and their analogues) are important dietary components which help regulate serum lipid profile, reduce total- and LDL-cholesterol
levels, and increase HDL/LDL ratio. In addition, plant sterols
possess anticancer, anti-inflammatory, and moderate antioxidant activities [29]. For instance, β-sitosterol, the most
abundant plant sterol in the human diet, displays significant effects on reducing the symptoms of benign prostatic
hyperplasia and prostate cancer. Moreover, this compound
has been associated with antidiabetic, immunomodulatory,
and analgesic properties [30]. Phytosterols are found
abundantly in nonpolar fractions of plants, and their daily
consumption is estimated in the range of 200–400 mg with
the main dietary sources being vegetable oils, nuts, cereal
products, vegetables, fruits, and berries [30]. They are also
known to be present in abundance in the fruits derived
from numerous genera of Rosaceae, including Prunus,
Crataegus, and Rosa [25]. In the lipid fraction of rosaceous
fruits, β-sitosterol was often identified as the predominant
lipophilic compound, constituting usually more than 60%
of the total sterols. As the daily intake of phytosterols
(1.5–2.4 g) required for beneficial health effects, especially
for cardiovascular and antiatherogenic protection, is usually higher than consumed with the common diet [30],
dietary supplementation is a rational solution, and new plant
sources of these biomolecules, such as the Cotoneaster fruits,
offer promise in this aspect.
3.3. Polyphenolic Profiling of Fruit Extracts. LC-MS analysis
of the hydrophilic (70% aqueous methanolic) extracts of the
Cotoneaster fruits revealed the presence of a number of
8
Oxidative Medicine and Cellular Longevity
Intens.
(mAU)
Cotoneaster bullatus O_RA8_01_943.d: UV chromatogram, 280 nm
12
15
2
3
4
10
7
5
1
8
6
14
10
16
11
18
5 6
2
1
10
7
4
8
10
14
12
19
17
15
16
11
9
18
26
C. bullatus
25
20 9_GB1_01_257.d: UV chromatogram, 280 nm
26
21
C. splendens
25
22
23
13
24
5
Intens.
(mAU)
21
22
23
24
13
5
0
Intens.
(mAU)
15
17 19 20
15
0
2
15
3
1
7
9
8
4
10
10
5 6
Cotoneaster hjelmqvistii O_RB2_01_945.d: UV chromatogram, 280 nm
14
11
15
12
16
17 18
13
5
26
21 22
23
24
C. hjelmqvistii
25
20
19
0
0
5
10
15
20
Time (min)
25
30
35
Figure 3: Representative UHPLC-UV chromatograms of the C. bullatus, C. splendens, and C. hjelmqvistii fruit polar extracts (λ = 280 nm).
The peak numbers refer to those applied in Table 3.
polyphenols (UHPLC peaks 1–26, Figure 3, Table 3) that
were fully or tentatively identified by comparison of their
chromatographic behavior and ESI-MS3 fragmentation pattern with authentic standards or literature values. Three
major groups of polyphenols were recognized, including phenolic acids (3, 7, and 8) and their derivatives (1, 4, 5, and 11),
flavan-3-ols including proanthocyanidins (9, 10, 12–16, 18,
and 24), and flavonoids (17, 20, 21–23, 25, and 26). The
recorded UHPLC fingerprints (Table 3) indicate that the
phenolic profiles of all nine Cotoneaster fruits were qualitatively similar. However, noticeable differences were found
in the proportions of individual polyphenols, which allowed
the subgroups of species to be distinguished depending on
the prevalent phenolic class. A distinctive feature of most
Cotoneaster samples, especially C. divaricatus, C. horizontalis, and C. nanshan, was the predominance of phenolic acid
derivatives (1, 3–5, 7, 8, and 11), mainly caffeoylquinic acids,
with the dominant peak being chlorogenic acid (7). On the
other hand, C. zabelii, C. bullatus, and C. hjelmqvistii
contained relatively high amounts of flavan-3-ols and
proanthocyanidins (9, 10, 12–16, 18, and 24), with dominating (−)-epicatechin (12). The contribution of flavonoids
(17, 20, 21–23, 25, and 26) to the overall phenolic fraction
was generally the lowest, but C. splendens was distinguished by a particularly large proportion of quercetin
3-(2″-xylosyl)-galactoside (17), and C. dielsianus contained
a relatively higher level of hyperoside (21).
This report is the first comprehensive study of the
LC-MS characteristics of the Cotoneaster fruits; the previous
studies on C. integerrimus and C. pannosus have focused
only on a selected aspect (HPLC-PDA) of their polyphenolic profiles [9, 12]. In contrast to the present results,
the occurrence of low-molecular phenolic acids, including shikimic, p-coumaric, and benzoic acids, has been previously reported, and this phenomenon may be explained by
the individual attributes of the tested samples or by differences in the methodology employed for the structural identification. On the other hand, the reported high level of
(−)-epicatechin in the fruits of C. integerrimus [12] indicates its similarity to those of C. zabelii and C. bullatus
analyzed in the present study.
The total phenolic content (TPC) of the 70% aqueous
methanolic extracts of the Cotoneaster fruits was determined
by the Folin-Ciocalteu photometric assay, commonly used to
estimate phenolic metabolites as gallic acid equivalents
(GAE). As shown in Table 4 and Figure 2, the TPC values
in the analyzed fruits varied from 26.0 to 43.5 mg GAE/g of
fruit dw. The highest phenolic content was found for the fruits
of C. hjelmqvistii and C. zabelii (43.5 and 43.0 mg/g dw,
respectively), followed by those of C. splendens and C. bullatus
(38.5 and 37.3 mg/g dw, respectively). The level of phenolics
in these species is comparable with those observed for other
Rosaceae fruits reported in the literature as rich sources
of natural polyphenols, for example, Aronia melanocarpa
(Michx.) Elliott (34.4–78.5 mg GAE/g dw; [3]) and Sorbus
species (22.4–29.8 mg GAE/g dw; [16]).
The presence of polyphenolic compounds in fruits and
vegetables is strongly linked with the beneficial effects of
these food products for human health, and the influence of
polyphenols on closely intertwined processes of inflammation and oxidative stress is recognized as the most feasible
mode of this action. As free radical scavengers, metal chelators, prooxidant and proinflammatory enzyme inhibitors,
and modifiers of cell signaling pathways, polyphenols are
Compounds
tR
UV
(M-H)−m/z
MS/MS m/z
(% base peak)
1
Vanillic acid-hexoside
3.5
250, 290
329
MS2: 167 (100);
123 (2); 107 (4)
2
Unidentified
4.4
250, 295
255
3
3-O-Caffeoylquinic acid
6.0
294, 325
353
Number
4
3-O-p-Coumaroylquinic
acid
MS2: 165 (23)
MS2: 191 (100);
179 (47); 135 (6)
CL
CDV
CHR
CN
CH
%b
CDL
CS
CB
CZ
3.8
3.1
3.4
3.4
2.5
1.4
2.3
0.9
1.7
33.9
3.1
2.1
16.1
5.7
5.0
2.6
3.1
3.2
1.8
10.4
5.3
15.1
5.1
5.1
8.1
5.7
1.9
2.1
5.0
4.0
2.3
1.9
2.4
3.7
3.3
2.5
2
9.4
285, 310
337
MS : 163 (100);
119 (10)
2
5
Caffeic acid hexoside
9.8
290, 323
341
MS : 179 (100);
135 (10)
2.3
8.2
2.7
5.1
1.2
0.6
2.5
1.0
2.8
6
Unidentified
10.0
285, 323
439
MS2: 391 (100);
338 (17); 243 (10);
195 (55)
3.9
1.9
4.3
4.8
2.3
1.2
2.0
1.3
1.3
7
5-O-Caffeoylquinic acid
(chlorogenic acid)a
10.4
294, 325
353
MS2: 191 (100);
179 (6)
29.4
28.3
29.5
26.0
23.5
23.0
17.9
17.3
10.8
8
4-O-Caffeoylquinic acid
10.9
294, 325
353
1.1
4.1
2.4
5.1
1.0
1.4
2.4
2.1
2.0
9
Procyanidin B-type
dimer
13.7
280
577
0.7
1.6
0.5
1.2
1.1
1.2
0.9
nd
0.7
0.3
2.5
4.6
0.8
8.0
6.2
5.5
9.3
10.2
11.7
3.1
1.2
3.6
1.2
0.6
0.8
1.4
1.9
2.4
4.4
8.5
2.1
15.8
12.3
9.7
18.6
34.4
nd
nd
nd
nd
0.4
nd
0.6
0.9
1.2
nd
nd
nd
nd
1.0
nd
0.6
1.1
1.4
MS2: 191 (21); 179
(47); 173 (100)
MS2: 451 (30); 425
(100); 407 (55);
289 (10)
MS3 (425): 407
(80);
273 (13)
10
Procyanidin B-2a
14.9
280
577
MS2: 451 (25); 425
(100); 407 (62);
289 (14);
MS3 (425): 407
(95); 273 (9)
11
5-O-p-Coumaroylquinic
acid
15.7
285, 310
337
MS2: 191 (100);
163 (7)
Oxidative Medicine and Cellular Longevity
Table 3: UHPLC-PDA-ESI-MS3 data of polyphenols identified in the polar extracts from Cotoneaster fruits.
2
12
(−)-Epicatechina
16.4
280
289
13
Procyanidin B-type
dimer
17.3
280
577
14
Procyanidin B-type
tetramer
18.3
280
1153
MS : 245 (100);
205 (28)
MS2: 451 (25); 425
(100); 407 (45);
289 (6);
MS3 (425): 407
(75); 273 (9)
MS2: 1027 (15);
863 (80); 739 (15);
501 (05); 491 (58);
289 (100)
9
10
Table 3: Continued.
Number
Compounds
tR
UV
(M-H)−m/z
MS/MS m/z
(% base peak)
MS2: 847 (19); 739
(77); 713 (51); 695
(100); 577 (26);
MS3 (713): 695
(100); 561 (30);
543 (31); 425 (32);
407 (36)
MS2: 863 (90); 739
(10); 501 (65); 491
(62); 289 (100)
MS2: 463 (10); 445
(14); 300 (85);
MS3 (463): 343
(62); 301 (100)
CL
CDV
CHR
CN
CH
%b
CDL
CS
CB
CZ
0.5
1.9
3.2
nd
5.3
3.7
3.3
5.6
7.3
nd
nd
2.1
nd
2.7
2.2
2.0
2.7
3.5
nd
nd
4.0
nd
2.0
2.6
16.1
5.9
nd
nd
2.2
2.0
nd
1.1
nd
1.5
1.2
nd
nd
2.5
2.6
2.4
1.5
2.0
2.0
1.2
1.2
Procyanidin C-1a
20.6
280
865
16
Procyanidin B-type
tetramer
23.3
280
1153
17
Quercetin 3-O-β-D-(2″
-O-β-Dxylosyl)galactosidea
23.9
268, 355
595
18
Epicatechin derivative
26.2
280
739
19
Unidentified
26.3
280
451
20
Quercetin rhamnosidehexoside
26.7
275, 350
609
MS2: 301 (100)
0.6
0.4
1.4
nd
0.7
3.2
2.3
0.9
1.8
27.1
265, 355
463
MS2: 301 (100)
2.5
5.0
4.9
5.5
5.5
9.5
5.2
6.6
2.4
27.3
260, 355
609
MS2: 301 (100)
0.8
2.5
2.6
2.8
3.8
2.5
2.0
nd
2.2
28.0
265, 355
463
MS2: 301 (100)
1.6
3.1
2.4
2.4
3.5
2.5
3.3
2.6
3.2
21
22
23
24
Quercetin 3-O-β-Dgalactoside (hyperoside)a
Querectin
3-O-β-D-(6″-O-α-LRhamnosyl)glucoside
(rutin)a
Quercetin 3-O-β-Dglucoside (isoquercitrin)a
Procyanidin B-type
dimer
MS2: 587 (100);
451 (19); 339 (40);
289 (35)
MS2: 341 (100);
217 (8)
2
28.6
280
577
MS : 425 (100);
407 (52); 289 (18)
0.6
1.6
2.0
1.0
1.6
2.0
1.6
2.0
2.4
25
Quercetin rhamnosidehexoside
31.3
276, 350
609
MS2: 301 (100)
nd
2.2
2.2
nd
0.4
4.1
nd
2.9
nd
26
Quercetin 3-O-α-Lrhamnoside (quercitrin)a
32.4
276, 350
447
MS2: 301 (100)
nd
2.8
2.2
nd
1.4
5.1
1.8
2.6
nd
a
Identified with the corresponding standards; brelative contribution based on peak area on the UHPLC chromatograms (λ = 280 nm) recorded at the extract concentration of 10 mg/mL and injection volume of 3 μL;
nd—not detected; the values are means (n = 3); with RSD ≤ 5%. CL, C. lucidus; CDV, C. divaricatus; CHR, C. horizontalis; CN, C. nanshan; CH, C. hjelmqvistii; CDL, C. dielsianus; CS, C. splendens; CB, C. bullatus;
CZ, C. zabelii.
Oxidative Medicine and Cellular Longevity
15
Oxidative Medicine and Cellular Longevity
11
Table 4: Total phenolic content (TPC) and antioxidant activity (DPPH, FRAP, and TBARS tests) of the Cotoneaster fruits and standard
antioxidants.
Fruit sample/
standard
TPCa
(mg GAE/g)
C. lucidus
C. divaricatus
C. horizontalis
C. nanshan
C. hjelmqvistii
C. dielsianus
C. splendens
C. bullatus
C. zabelii
QU
BHA
BHT
TX
28.70 ± 1.01B
29.71 ± 0.91B
30.50 ± 0.72B
26.02 ± 0.74A
43.50 ± 1.21D
31.02 ± 1.02B
38.51 ± 0.81C
37.31 ± 0.80C
43.02 ± 1.11D
—
—
—
—
Radical scavenging activity
DPPHb
EC50
TE
(μg/mL)
(μmol TE/g)
123.41 ± 1.70E
91.47 ± 2.01C
93.32 ± 1.90C
178.35 ± 2.81F
64.51 ± 0.84B
117.10 ± 2.40D
67.15 ± 1.80B
66.31 ± 1.70B
62.93 ± 1.91B
1.70 ± 0.11A
2.90 ± 0.15A
6.50 ± 0.13A
3.80 ± 0.20A
Reducing powerc
FRAP
(mmol Fe2+/g)
TE
(μmol TE/g)
122.75 ± 1.69C 0.70 ± 0.01B
257.22 ± 4.96B,C
D
C
165.58 ± 3.62
0.76 ± 0.01
281.61 ± 4.43C
162.38 ± 3.31D 0.85 ± 0.01D
322.75 ± 4.06D
B
A
84.91 ± 1.33
0.61 ± 0.01
213.41 ± 4.42A
E,F
F
234.84 ± 2.91
1.05 ± 0.02
414.38 ± 11.14F,G
C
B
129.37 ± 2.65
0.67 ± 0.03
240.90 ± 13.83A,B
225.49 ± 6.04E
0.98 ± 0.01E
383.06 ± 6.24E,F
E
E
228.54 ± 5.86
0.97 ± 0.01
378.87 ± 2.90E
F
G
240.93 ± 7.28
1.09 ± 0.04
434.27 ± 20.50G
A
K
8.96 ± 0.58
31.20 ± 0.98 11878.15 ± 15.20J
5.24 ± 0.27A
16.14 ± 0.77I 7726.31 ± 10.52H
A
2.34 ± 0.05
18.89 ± 0.45J 9247.66 ± 12.30I
—
9.34 ± 0.35H
—
LA-peroxidation TBARSd
IC50
(μg/mL)
TE
(μmol TE/g)
108.70 ± 4.11F
83.16 ± 0.58D
84.89 ± 2.11D
165.76 ± 3.74G
62.96 ± 1.10C
103.72 ± 2.58E
66.21 ± 2.94C
64.99 ± 1.55C
62.54 ± 1.32C
1.85 ± 0.12A
3.16 ± 0.22A
9.31 ± 0.16B
8.47 ± 0.45B
314.84 ± 6.03C
406.94 ± 1.43D
401.23 ± 5.03D
205.30 ± 2.33B
532.92 ± 4.63E,F
322.66 ± 3.98C
518.18 ± 11.79E
523.90 ± 6.30E,F
543.86 ± 5.76F
18.37 ± 1.69A
10.76 ± 1.06A
3.64 ± 0.09A
—
a–d
Results expressed as means ± SD calculated per dw of the plant material (n = 3); different capital letters within the same row indicate significant differences at
α = 0 05 in HSD Tukey’s test. aTotal phenolic content (TPC), expressed in gallic acid equivalents (GAE). bScavenging efficiency in the DPPH test, the amount of
the plant materials or standards required for 50% reduction of the initial DPPH concentration expressed as EC50, effective concentration. cFerric reducing
antioxidant power. dAbility to inhibit linoleic acid (LA) peroxidation monitored by TBARS test and expressed as IC50, concentration of plant materials or
standards needed to decrease the LA-peroxidation by 50%; TE, Trolox® equivalent antioxidant activity. Standards: QU, quercetin; BHA, butylated
hydroxyanisole; BHT, 2,6-di-tert-butyl-4-methylphenol; TX, Trolox®.
effective agents preventing damages related to the oxidative
stress and inflammation implicated in the etiology and
progression of numerous chronic diseases, including cardiovascular diseases, diabetes mellitus, neurodegenerative disorders, and cancer [31–33]. The occurrence of polyphenolic
compounds in the investigated fruits might thus largely
define their bioactivity, especially that Cotoneaster-derived
polyphenols have been previously linked with strong antioxidant capacity in our earlier study regarding the leaves [34].
3.4. Biological Activity. The above presented phytochemical
studies proved that fruits of Cotoneaster species are indeed
a rich source of diverse phytochemicals with a wide spectrum
of recognized biological properties. However, based on the
results of the quantitative studies, the polyphenolic fraction with the highest content would appear to have the
greatest beneficial health effects of the fruits in a human
organism. Thus, further studies were focused on providing
a more detailed insight into potential mechanisms of the
activity of the hydrophilic components, that is, their antiinflammatory and antioxidant effects.
3.4.1. Inhibitory Effects on Two Enzymes Involved in
Inflammation. Inflammation is a complex process that constitutes a part of the immune system defense against harmful
stimuli, but may lead to negative effects if uncontrolled. The
inflammatory response is regulated by numerous enzymes
and mediators and thus can be intercepted at different points,
and several of these key enzymes, including lipoxygenases
(LOX) and hyaluronidases (HYAL), are most often used
to determine the anti-inflammatory potential of natural
products [35]. LOX catalyze the diooxygenation of arachidonic acid to form hydroperoxides, the first step in the
biosynthesis of several proinflammatory mediators [36].
HYAL, on the other hand, are highly specific hydrolases
that degrade hyaluronic acid, an important component of
the extracellular matrix, thus increasing the permeability
of the tissues and facilitating the spread of inflammation
[37]. Our present findings indicate that all fruit extracts
inhibit the activity of LOX and HYAL in a dosedependent manner (Table 5). The strongest inhibitory effect
towards LOX was demonstrated by the leaf extracts of
C. hjelmqvistii and C. zabelii (IC50 = 7.70 and 9.97 μg/U,
respectively), while the activity of HYAL was most strongly
hindered by the leaf extract of C. lucidus (IC50 = 16.44 μg/U).
The activity of the extracts was weaker in comparison to
indomethacin (IC50 = 1.89 μg/U for LOX and 5.60 μg/U
for HYAL), but after recalculating the results to adjust
for the actual polyphenol content (which gives IC50 values
in the range of 0.33–0.77 μg/U for LOX and 0.47–1.93 μg/
U for HYAL inhibition), the activity of the extracts looks
quite advantageous in comparison to the positive standard.
The anti-inflammatory potential of Cotoneaster polyphenols
is further confirmed by the high activity of (−)-epicatechin,
quercetin, and chlorogenic acid, the main constituents of
the investigated leaf extracts.
3.4.2. Antioxidant Activity in Chemical Models. The basic
antioxidant mechanism of Cotoneaster polyphenols was verified in chemical models using three complementary in vitro
assays: DPPH and FRAP tests, two of the most frequently
12
Oxidative Medicine and Cellular Longevity
Table 5: Inhibitory effects of Cotoneaster fruit extracts and standards towards lipoxygenase (LOX) and hyaluronidase (HYAL).
LOX
Fruit sample/standard
C. lucidus
C. divaricatus
C. horizontalis
C. nanshan
C. hjelmqvistii
C. dielsianus
C. splendens
C. bullatus
C. zabelii
QU
ECA
CHA
IND
HYAL
IC50a
(μg/mL)
IC50b
(μg/U)
IC50a
(μg/mL)
IC50b
(μg/U)
487.75 ± 6.57F
479.98 ± 12.79F
421.85 ± 5.78E
626.16 ± 5.04H
290 ± 2.75C
914.97 ± 2.15J
734.25 ± 5.86I
585.43 ± 16.14G
375.87 ± 9.89D
69.60 ± 2.62A
124.38 ± 1.56B
151.71 ± 7.52B
90.12 ± 0.40A
13.29 ± 0.18F
13.08 ± 0.35F
11.50 ± 0.16E
17.07 ± 0.14H
7.70 ± 0.07C
24.94 ± 0.06J
20.01 ± 0.16I
15.96 ± 0.44G
9.97 ± 0.26D
2.46 ± 0.01A
3.39 ± 0.04B
4.14 ± 0.21B
1.89 ± 0.10A
25.65 ± 0.95C
34.22 ± 1.48D
40.51 ± 2.11E,F,G
45.64 ± 0.76G
44.44 ± 1.72F,G
35.07 ± 2.60D,E
34.36 ± 0.11D
39.04 ± 0.82D,E,F
33.33 ± 2.12D
21.04 ± 1.03C
18.51 ± 0.50B
20.35 ± 0.36B
8.61 ± 0.22A
16.44 ± 0.61C
21.93 ± 0.95D
25.97 ± 1.35E,F,G
29.25 ± 0.49G
28.48 ± 1.10F,G
22.48 ± 1.66D,E
22.03 ± 0.07D
25.03 ± 0.53D,E,F
21.37 ± 1.36D
13.87 ± 0.06C
11.87 ± 0.32B
13.05 ± 0.23B
5.60 ± 0.07A
Results expressed as means ± SD calculated per dry weight (dw) of the extracts; different capital letters within the same row indicate significant differences at
α = 0 05 in HSD Tukey’s test. Standards: QU, quercetin; ECA, (−)-epicatechin; CHA, chlorogenic acid; IND, indomethacin. Ability to inhibit lipoxygenase
(LOX) and hyaluronidase (HYAL) calculated as the amount of analyte needed for 50% inhibition of enzyme activity was expressed as follows: aμg of the dry
extracts or standards/mL of the enzyme solution and bμg of the extracts/enzyme units (U).
employed SET (single electron transfer) type methods, and
the inhibition of AAPH-induced linoleic acid peroxidation
test (monitored by TBARS assay), a more physiologically relevant system which involves the HAT (hydrogen atom transfer) mechanism. In all of the applied tests, the investigated
fruits displayed concentration-dependent activity with the
capacity parameters (expressed in μmol TE/g dw) of a similar
order of magnitude, which shows that Cotoneaster antioxidants can effectively act via both basic mechanisms. The
highest activity in comparison to the natural (quercetin)
and synthetic standards (BHA and BHT) were observed in
the FRAP and TBARS assays for all fruits (Table 4 and
Figure 2). In all tests, the fruits of C. zabelii, C. hjelmqvistii,
C. bullatus, and C. splendens, indicated in the present
study as the richest sources of polyphenols, displayed the
highest antioxidant efficiency, with the activity parameters
varying in the narrow range of 225.5–240.9 μmol TE/g dw
(DPPH), 378.9–434.3 μmol TE/g (FRAP), and 518.2–
543.9 μmol TE/g (TBARS), respectively. Interestingly, these
were the species that also exhibited the relatively largest
proportions of proanthocyanidins/flavan-3-ols (C. zabelii,
C. bullatus, C. splendens) or quercetin 3-(2″-xylosyl)-glucoside (C. hjelmqvistii), which suggest that these polyphenols
play a significant role in the activity of fruits. Additionally,
the close connection between the phenolic levels and antioxidant parameters was also evidenced by statistically significant linear correlations between TPCs and the results
of the DPPH (∣r∣ = 0 9352, p < 0 001), FRAP (∣r∣ = 0 9491,
p < 0 001), and TBARS (∣r∣ = 0 9116, p < 0 001) tests.
3.4.3. Protective Effects on Human Plasma Components
Exposed to Oxidative Stress. To provide a more detailed
insight into the antioxidant effects of Cotoneaster polyphenols, the four most promising species (C. zabelii, C. bullatus,
C. splendens, and C. hjelmqvistii) were selected for further
studies in a biological model. Since according to traditional
application and our present results, Cotoneaster fruits appear
to be promising sources of phytochemicals with properties
especially advantageous for the circulatory system (i.e., linoleic acid and β-sitosterol), a human plasma model was
selected to evaluate their additional benefits for cardiovascular health, this time mediated by polyphenols. This approach
allowed for the in vitro monitoring of the protective effects of
the extracts towards human plasma components under
oxidative stress conditions. The peroxynitrite (ONOO−) used
for inducing oxidative stress is a known in vivo-operating
oxidant, responsible for structural changes in plasma proteins and lipids and implicated in numerous oxidative
stress-related disorders [38]. The concentrations of ONOO−
(100 and 150 μM) selected for the study enabled quantitative
measurements of the resulting modifications in plasma components, but may be also regarded as physiologically-relevant
as they can be reached in vivo in local compartments, for
example, during a serious inflammation of blood vessels [39].
The addition of ONOO− to the plasma samples resulted
in an overall decrease (p < 0 001) in the nonenzymatic antioxidant capacity of the plasma, measured as the FRAP
parameter, and in oxidative and nitrative alterations of its
protein and lipid components, which was evidenced by a
significant increase (p < 0 001) in lipid peroxidation biomarkers (lipid hydroperoxides and TBARS), a noticeable rise
(p < 0 001) in 3-nitrotyrosine level (marker of protein nitration), and a decrease (p < 0 001) in the level of thiol groups
(marker of protein oxidation). On the other hand, in the
plasma samples incubated with ONOO− in the presence of
Cotoneaster extracts (1–50 μg/mL), the extent of oxidative/
nitrative damage to both proteins and lipids was noticeably
limited (p < 0 05), regardless of the tested species and the
Oxidative Medicine and Cellular Longevity
13
0.50
3NT-Fg
–SH groups
−SH groups (휇mol/mL of plasma)
⁎⁎⁎⁎⁎⁎
1.20
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎⁎⁎⁎
⁎⁎⁎
0.80
0.40
1 5 50 1
CB
5 50 1
5 50 1 5 50
CS
CH
⁎⁎⁎
⁎⁎⁎⁎⁎⁎ ⁎⁎⁎
0.30
###
0.10
0.00
5 휇g/mL
CZ
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎⁎⁎⁎⁎⁎⁎⁎⁎⁎⁎⁎⁎
⁎⁎⁎⁎⁎⁎⁎⁎⁎
⁎⁎⁎
0.20
RT
TX
ECA
Control
ONOO−
0.00
0.40
1 5 50 1 5 50 1
CH
CB
Extract concentration (휇g/mL)
(a)
CZ
5 휇g/mL
(b)
0.15
###
TBARS (휇mol/mL of plasma)
0.60
⁎⁎⁎⁎⁎⁎
⁎⁎⁎⁎⁎⁎ ⁎⁎⁎⁎⁎⁎⁎⁎⁎⁎⁎⁎
⁎⁎⁎
⁎⁎⁎⁎⁎⁎⁎⁎⁎ ⁎⁎⁎⁎⁎⁎⁎⁎⁎
⁎⁎⁎
0.40
CS
CH
5 50
⁎⁎⁎⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎⁎⁎⁎ ⁎⁎⁎⁎⁎⁎⁎⁎⁎⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
0.06
0.03
5 휇g/mL
CZ
⁎⁎⁎
⁎⁎⁎⁎⁎⁎
0.09
0.00
ECA
5 50 1
0.12
1 5 50 1 5 50 1 5 50 1 5 50
Control
ONOO−
CB
5 50 1
TX
5 50 1
CHA
1
RT
0.20
Control
TBARS
LOOH
CB
CS
CH
Extract concentration (휇g/mL)
CZ
CHA
RT
TX
ECA
###
0.80
ONOO−
LOOH (nmol/mg of plasma protein)
CS
Extract concentration (휇g/mL)
1.00
0.00
5 50 1 5 50
CHA
RT
TX
ECA
1.60
Control
ONOO−
###
CHA
3-NT-Fg (nmol/mg of plasma protein)
2.00
5 휇g/mL
Extract concentration (휇g/mL)
(c)
(d)
0.60
FRAP
⁎⁎⁎
Fe2+ (mM)
0.45
⁎⁎⁎⁎⁎⁎⁎⁎⁎ ⁎⁎⁎
⁎⁎⁎
⁎⁎⁎⁎⁎⁎ ⁎⁎⁎
⁎⁎⁎ ⁎⁎⁎
⁎⁎⁎ ⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
###
0.30
CB
5 50 1
CS
CH
5 50
CZ
ECA
5 50 1
RT
5 50 1
TX
1
CHA
Control
0.00
ONOO−
0.15
5 휇g/mL
Extract concentration (휇g/mL)
(e)
Figure 4: Effects of the Cotoneaster fruit extracts on human plasma exposed to oxidative stress: (a) effects on the nitration of tyrosine residues in
plasma proteins and formation of 3-nitrotyrosine (3-NT-Fg); (b) effects on the oxidation of free thiol groups (−SH); effects on the peroxidation of
plasma lipids including (c) formation of lipid hydroperoxides (LOOH), and (d) thiobarbituric acid-reactive substances (TBARS); (e) effects on
ferric reducing ability of blood plasma (FRAP). Results expressed as means ± SE (n = 8) for repeated measures: ### p < 0 001, for ONOO−-treated
plasma (without the extracts) versus control plasma, and ∗∗∗ p < 0 001 for plasma treated with ONOO− in the presence of the investigated extracts
(1–50 μg/mL) or the standards (5 μg/mL). CB, C. bullatus; CH, C. hjelmqvistii; CS, C. splendens; CZ, C. zabelii. Standards: CHA, chlorogenic
acid; RT, rutin; TX, Trolox®; ECA, (−)-epicatechin.
extract concentration. As shown in Figures 4(a) and 4(b),
even at the lowest concentrations of 1 μg/mL, the extracts
were able to reduce tyrosine nitration by about 29–42%
and thiol group oxidation by about 24–26%, while at the
concentration of 50 μg/mL the effectiveness rose to 46–55%
and 29–32%, respectively. Moreover, as demonstrated in
Figures 4(c) and 4(d), all fruit samples inhibited the generation of plasma lipid hydroperoxides by 40–50% and reduced
14
TBARS levels by 19–35%. All extract-treated samples, apart
from those fortified with 1 μg/mL of C. bullatus extract, demonstrated a statistically significant (p < 0 001) improvement
in the nonenzymatic antioxidant capacity of blood plasma
of up to 44% in comparison to the samples not protected
by the extracts (Figure 4(e)). In most cases, little difference
was observed in the activity between the tested fruits;
however, the inhibition of tyrosine nitration assay found
C. bullatus and C. zabelii displaying stronger activity than the
other two extracts at all concentrations tested (p < 0 05). A
dose dependency was noticeable for C. bullatus and C.
splendens in antinitrative activity (Figure 4(a)) and for most
Cotoneaster species in the TBARS test, with the exception
of C. zabelii (Figure 4(d)). Some significant correlations were
also found, between the TPCs and the activity parameters.
The most prominent was the relationship for the FRAP assay
(∣r∣ = 0 7587, p < 0 01). In the tests for protein protection, the
correlation between the percentage inhibition of tyrosine
nitration and phenolic level was stronger (∣r∣ = 0 6774,
p < 0 05) than the analogous relationship for the reduction
of thiol group oxidation (∣r∣ = 0 4885, p < 0 05). Contrastingly, the correlations in the lipid peroxidation assays were
not statistically significant (p > 0 05).
The effectiveness of the extracts was further supported by
the fact that in all of the tests, the observed antioxidant effects
of the fruit extracts at the corresponding concentration levels
(5 μg/mL) were similar or higher to that of Trolox®, a synthetic analog of vitamin E often used as a positive standard
in antioxidant studies. Moreover, the significant activity of
rutin, chlorogenic acid, and, especially, (−)-epicatechin confirm the important role of polyphenols in the capacity of
the extracts.
The wide range of the extract concentrations tested
(1–50 μg/mL) was in accordance with the general practice
of in vitro studies [20] and allowed for the study of different
interactions in the system. Additionally, the lower levels
(1–5 μg/mL) might be considered physiologically-relevant
as they correspond to the levels of phenolics attainable
in vivo after consumption of polyphenol-rich plant materials. For example, according to the accumulated research
[40, 41], the maximal achievable concentration of plant
phenolics in blood plasma can reach up to 5–10 μM,
which generally corresponds to less than 5 μg/mL. Taking
into account the TPC levels evaluated for Cotoneaster
fruits in the present study and the extraction efficiency
(15–30%, depending on the species), the levels of phenolics corresponding to the applied extract concentration of
1–5 μg/mL are about 0.13–1.25 μg/mL: well within the
obtainable plasma range. This suggests that the protective
activity of the Cotoneaster extracts towards ONOO−induced changes observed in vitro may translate to their
positive in vivo effects.
The harmful influence of ONOO− is often associated with
serious pathological consequences in many organs and systems of the human body. The nitration/oxidation of biomolecules such as enzymes, receptors, lipoproteins, fatty acids, or
nucleic acids changes their function and may impair cellular
signalization pathways, induce inflammatory responses, or
even promote cell apoptosis [38, 39]. In the case of the
Oxidative Medicine and Cellular Longevity
circulatory system, the negative effects of ONOO− result in
a higher risk of cardiovascular disorders, such as stroke, myocardial infarction, or chronic heart failure [38], and are connected with the direct modifications of plasma proteins and
lipids. For instance, the formation of 3-nitrotyrosine in
fibrinogen might contribute to prothrombotic events in
the blood coagulation cascade and fibrinolysis process
[42], while thiol oxidation in platelet proteins leads to the
inhibition of platelet function [43]. Additionally, oxidation
of low-molecular-weight thiols, such as reduced glutathione, diminishes the endogenous antioxidant capacity of
plasma and primes further oxidative damage in the system
[38]. Similarly, lipid peroxidation initiated by ONOO− may
propagate platelet aggregation [44], while peroxynitritemodified LDL binds with high affinity to macrophage
scavenger receptors leading to foam cell formation, which
represent a key early event in atherogenesis [38, 45]. The
prevention of these processes partially explains the beneficial
effects of Cotoneaster fruits reported by traditional medicine
and might be regarded as a good strategy in prophylaxis of
various cardiovascular complaints.
3.5. Cellular Safety. Due to its long tradition of consumption
and application in folk medicine, the Cotoneaster fruits might
be regarded as nontoxic. However, in the case of the concentrated extracts, a more detailed evaluation of their safety is
required. Therefore, the next step of our research was a viability test on PMBCs which assessed the cytotoxicity of the
extracts. After two, four, and six-hour incubation periods
with the plant extracts at concentrations of 5, 25, and
50 μg/mL, the viability of the extract-treated cells constituted
97.3–101.7% of that of the control (non-treated cells) and no
statistically significant differences were found (p > 0 05)
between the two values (Figure 5). These findings suggest
that the Cotoneaster extracts do not have cytotoxic effects at
these concentrations.
4. Conclusion
The current paper presents the first comprehensive phytochemical and activity study of Cotoneaster fruits. The fruits
were found to possess distinct lipophilic and phenolic profiles, significant antioxidant activity in both chemical and
biological models, noticeable inhibitory effects on the proinflammatory enzymes, and cellular safety. Hence, Cotoneaster
fruits appear to be promising candidates for the production
of pharma- and nutraceuticals associated with preventing
and treating oxidative stress and inflammatory-related
chronic diseases; they may also contribute to a balanced
and varied diet comprising food rich in bioactive compounds.
Furthermore, the protective effects against ONOO−-induced
modifications in the plasma components, demonstrated by
the polyphenolic fractions from the fruits of C. hjelmqvistii,
C. zabelii, C. splendens, and C. bullatus at in vivo-relevant
levels, may be considered as a molecular basis for the beneficial effects of Cotoneaster fruits within the cardiovascular system reported by traditional medicine. The biological activity
demonstrated in the present study might therefore be a starting point of more extensive investigation on the nutritional
Oxidative Medicine and Cellular Longevity
15
PMBC viability
120
% of viability
100
80
60
40
20
0
Control
5
25
50
C. bullatus
5
25
C. hjelmqvistii
50
5
25
C. splendens
50
5
25
50
C. zabelii
Extract concentration (휇g/mL)
2h
4h
6h
Figure 5: Viability of peripheral blood mononuclear cells (PMBCs) after 2, 4, and 6 h incubation with the Cotoneaster fruit extracts at 5, 25,
and 50 μg/mL. Results are presented as means ± SD (n = 14).
value and bioactivity of Cotoneaster fruits, including their
effects in in vivo systems.
Data Availability
The data used to support the findings of this study are
available from the corresponding author upon request.
Conflicts of Interest
The authors report no conflicts of interest.
Acknowledgments
This work was financially supported by the University of
Lodz, Poland through Grant no. 506/1136 (the synthesis of
peroxynitrite, ONOO−), the Medical University of Lodz,
Poland through Grant no. 503/3-022-01/503-31-001 (the
phytochemical and anti-inflammatory activity studies), and
the National Science Centre, Poland through Grant no.
2017/01/X/NZ7/00520 (all other costs). The authors would
like to thank the staff of the Botanical Garden in Lodz and
the Forestry Experimental Station of the Warsaw University
of Life Sciences in Rogow for providing and authenticating
the plant material.
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