Antifungal Polyamides of Hydroxycinnamic Acids from Sunflower Bee Pollen

Article abstract of DOI:10.1021/acs.jafc.8b03976

The aim of the bioassay-guided fractionation was the selection of the most potent group of compounds responsible for the protection of sunflower bee pollen grains. Synthesis of prospective antifungal polyamides of hydroxycinnamic acids was based on previo

Full text of DOI:10.1021/acs.jafc.8b03976

Article
pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Antifungal Polyamides of Hydroxycinnamic Acids from Sunflower
Bee Pollen
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Jan Kyselka,*,† Roman Bleha, Miroslav Dragoun, Kristyna Bialasova,‡ Sarka Horackova,
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Martin Schatz, Marcela Slukova, Vladimír Filip, and Andriy Synytsya
†Department of Dairy, Fat and Cosmetics, Faculty of Food and Biochemical Technology, Department of Carbohydrates and
Cereals, Faculty of Food and Biochemical Technology, and Department of Computing and Control Engineering, Faculty of
‡
§
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Chemical Engineering, University of Chemistry and Technology Prague, Technicka 5, Prague 166 28, Czech Republic
S
* Supporting Information
ABSTRACT: The aim of the bioassay-guided fractionation was the selection of the most potent group of compounds
responsible for the protection of sunflower bee pollen grains. Synthesis of prospective antifungal polyamides of
hydroxycinnamic acids was based on previous structural elucidation of ethanol soluble fraction by 1H,1H-PFG-COSY,
1H,13C-HSQC, FT-IR, FT-Raman, and LC−MS experiments. The main compounds found were tri-p-coumaroylspermidines
accompanied by other HCAA of spermidine and putrescine. Several model HCAA derivatives were prepared to test their
antifungal activity against widespread spoilage fungi (A. niger 42 CCM 8189, F. culmorum DMF 0103, and P. verrucosum DMF
0023). A. niger CCM 8189 and F. culmorum DMF 0103 exhibited higher resistance to the antifungal effects of hydroxycinnamic
acid amides, whereas P. verrucosum DMF 0023 was the most sensitive strain. It has been discovered the effect of HCAA polarity
on the role of secondary metabolites in the microbial protection of pollen grains. The combination of bioassay-guided
fractionation, structural elucidation, selection of prospective compounds, and their synthesis to determine their antifungal
properties could be considered as an original approach.
KEYWORDS: hydroxycinnamic acid amides, sunflower bee pollen, Helianthus annuus L., structural elucidation, antifungal activity
other mycotoxins under optimal growth conditions.5,7 Micro-
biological safety of bee pollen is dependent on the preservation
either by common microflora or by natural compounds derived
from plant pollen. Preservative phytochemicals are mainly
INTRODUCTION
Pollen is the male reproductive cells produced in the anthers of
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spermatophytes and plays an essential role in the life cycle of
flowering plants.1,2 Pollen grains are excellent sources of
expected to be responsible for the pollen protection against
carbohydrates, proteins, lipids, vitamins, and minerals neces-
sary for the plant growth, development, and fusion with a
fungal spoilage.8
female gamete.2,3 Moreover, nature has equipped pollen with
Suitable phytochemicals have to be located at the surface of
pollen grains, hydrophobic and widespread in the plant
several secondary metabolites to protect them against biotic
kingdom. Such requirements meet hydroxycinnamic acid
and abiotic stress. Phytochemical analysis has confirmed the
amides (HCAA), specific metabolites of pollen outer exine.
Their accumulation in the floral parts of plants affects flower
development, sexual differentiation, fertilization, and senes-
presence of carotenoids, phytosterols, flavonoids, and phenolic
acid derivatives.1,3,4 Plant pollens are gathered by worker
honeybees (Apis mellifera) and compacted into granules by
cence.9,10 Water-soluble aliphatic and aromatic amides of
hydroxycinnamic acids are widespread constituents of seeds,
roots, flowers, and female reproductive cells. Physiological
significance of less substituted amides is plant adaptation to
water stress, heat shock, UV radiation, sulfur starvation, and
deficiencies of K, Ca, Mg, and phosphorus.9,11 Infection of
Nicotiana tabacum leaves by tobacco mosaic virus (TMV)
induces accumulation of feruloyl and p-coumaroylputrescine in
necrotic lesions leading to limited multiplication of TMV
virus.12 Similarly, accumulation of feruloyl and p-coumaroyltyr-
amine upon wounding of potatoes is linked to the protection
against Phytophthora infestans and Streptomyces scabies.9,13
More substituted HCAAs of spermidine and putrescine are
plant nectar and saliva enzymes.2,5 After that, bee pollen is
transported to bee hive and used for the production of bee
bread or royal jelly to feed the larvae and drone bees.5,6
Apicultural products including honey, royal jelly, beeswax,
and bee pollen have been used in the human diet and personal
care for many thousands of years. Current growing demand for
bee pollen is connected with the consumption of natural food
supplements with antioxidant, hepatoprotective, or anti-
inflamatory benefits.1,6,7 However, bee pollen should be also
seen as nutrient-rich medium for the growth of fungi affecting
the shelf life and safety of food as the main quality criterions.
Aspergillus spp., Fusarium spp., Penicillium spp., Mucor spp.,
Alternaria spp., and Cladosporium spp. were the most frequent
isolates of molds identified in several bee pollen samples. They
are common saprophytes, which can be found either in the gut
of worker honeybees or in brood combs.5,7,8 Special attention
should be paid to Aspergillus, Fusarium, and Penicillium species
producing aflatoxins, ochratoxins, trichothecenes, and many
Received: July 26, 2018
Revised: October 4, 2018
Accepted: October 8, 2018
Published: October 8, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.8b03976
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
ether, from 100:0 to 87:13), Fr-2 (eluted with petroleum ether/
diethyl ether, from 87:13 to 0:100), and Fr-3 (eluted with acetone
and methanol). Each fraction containing compounds of the similar
polarity was tested for antifungal activity to identify the most
prominent compounds.
specific metabolites of pollen coat, where they might protect
pollen grains against plant diseases caused by microorganisms
and other results of damage, such as UV radiation and
oxidative stress.9−11
There are a number of publications focusing on the
biological activities of bee pollens and their extracts, even
though they contained thousands of bioactive compounds. The
present study reports the structural analysis of sunflower (
Helianthus annuus L.) bee pollen fractions focusing on close-up
composition of present hydroxycinnamic acid amides. The aim
of this work was to synthesize pure HCAAs of putrescine and
spermidine and to test their activity against widespread
spoilage fungi. Our conclusions may help to understand the
role of secondary metabolites in the microbial protection of
pollen grains.
Synthesis of p-Coumaric and Ferulic Acid Amides of
Putrescine. To a stirred solution of selected phenolic acid (9.0
mmol; 1.48 g of p-coumaric acid, 1.75 g of ferulic acid, respectively)
and N-Boc-1,4-butanediamine (8.5 mmol, 1.60 g) in anhydrous
CH2Cl2 (150 mL) on an ice water bath was added dropwise a
solution of N,N′-dicyclohexylcarbodiimide (DCC, 14.4 mmol, 2.97 g)
in 65 mL of CH2Cl2. The reaction mixture was allowed to warm to
laboratory temperature and stirred for 2 days until the reaction was
completed. Precipitated dicyclohexylurea was filtered off. Monoacy-
lated derivatives of N-Boc-1,4-butanediamine were purified by column
chromatography on silica gel (70−230 mesh). t-Butyl carbamate
group was deprotected by 30 mL of CF3COOH in CH2Cl2 (150 mL)
under an inert atmosphere of argon. After the mixture had been
stirred for 40 min at 25 °C, the solvent was evaporated under reduced
pressure. The residue was taken up in the mixture of methanol (25
mL) with hydrochloric acid (50 mL, 1 mol/dm3) and evaporated to
dryness. The last step was repeated two times. Finally, dissolution of
the resulting phenolamides in abs EtOH and subsequent evaporation
to dryness provided the desired products p-coumaroyl putrescine (1a)
and feruloyl putrescine (1b) in the form of hydrochloride.14 Di-p-
coumaroyl putrescine (2) was synthesized in the same manner with
the exception that amino groups were not protected by t-butyl
carbamate group. p-Coumaroyl putrescine and feruloyl putrescine
were obtained in 93% and 89% yields, respectively.
MATERIALS AND METHODS
■
Reagents and Instrumentation. Ferulic acid, p-coumaric acid,
putrescine, spermidine, N-Boc-1,4-butanediamine, N,N′-dicyclohex-
ylcarbodiimide, tetrahydrofuran, trifluoroacetic acid, and hydrochloric
acid were purchased from Sigma-Aldrich Co. All other reagents and
solvents were of analytical grade. FTIR spectra (spectral region 4000−
400 cm−1, 64 scans, resolution 2 cm−1) of the fractions were recorded
in the form of KBr tablets on Nicolet 6700 FTIR spectrometer
(Thermo Scientific, Waltham, MA) using Omnic 8.0 software. FT
Raman spectra (spectral region 4000−100 cm−1, 1000 scans,
resolution 4 cm−1) of the fractions were recorded with FT Raman
module of FTIR Nicolet iS50 spectrometer (ThermoScientific,
Waltham, MA), equipped with a Nd:YAG laser (excitation line
1064 nm, laser power 100 mW), CaF2 beam splitter, and InGaAs
detector. Obtained spectra were exported to Origin 6.0 (Microcal
Origin, U.S.) software in CSV or TXT format, where they were 5-
point filtered and baseline corrected. The second derivative algorithm
p-Coumaroyl Putrescine Hydrochloride (1a). The product was
obtained in 93% yield as a yellowish-brown solid. Mp = 102−104 °C;
Rf = 0.60 (CH2Cl2/CH3OH/27% aq NH3, 2:2:1); UV (CH3OH) λmax
308 nm; 1H NMR (500 MHz, CD3OD) δ 1.47 (4H, m, H-3, 4), 2.72
(2H, t, J = 7.2 Hz, H-2), 3.16 (2H, t, J = 6.5 Hz, H-5), 6.30 (1H, d, J
= 15.9 Hz, H-8′), 6.57 (2H, d, J = 8.5 Hz, H-3′, 5′), 7.21 (2H, d, J =
8.5 Hz, H-2′, 6′), 7.32 (1H, d, J = 15.9 Hz, H-7′); 13C NMR (126
MHz, CD3OD) δ 25.9 (C-4), 27.0 (C-3), 40.4 (C-2), 40.8 (C-5),
115.2 (C-8′), 117.0 (C-3′, 5′), 127.0 (C-1′), 131.3 (C-2′, 6′’), 144.7
(C-7′), 161.5 (C-4′), 168.7 (C-9′); IR (KBr) 3327, 1211 cm−1 (OH),
3066, 3036, 1167, 980 cm−1 (CH), 2929, 2851, 1448, 1440, 1371,
736 cm−1 (CH2), 1644, 1537, 1344 cm−1 (CONH), 1627, 1601,
1580, 1514 cm−1 (CC), 1132, 828 cm−1 (CNC), 1089, 1048 cm−1
(CC, CN), 641, 515 cm−1 (skeletal); Raman (λex 1064 nm) 3328,
1211 cm−1 (OH), 3066, 3036, 1168, 979 cm−1 (CH), 2935, 2854,
1444, 1347 cm−1 (CH2), 1706 cm−1 (CO), 1643, 1317 cm−1
(CONH), 1627, 1600, 1585, 1517 cm−1 (CC), 1131, 858 cm−1
(CNC), 1074, 1050, 1029 cm−1 (CC, CN), 644, 536, 455, 414, and
372 cm−1 (skeletal); HRESIMS m/z 235.14414 [M + H]+ (calcd for
was used for analysis of overlapped bands. H NMR and 13C NMR
1
APT spectra of the compounds or fractions dissolved in deuterated
solvents were recorded on Bruker Avance 600 and Bruker Avance 500
(Bruker, Inc., Billerica, MA) spectrometers. Working frequencies were
1
600.1 and 499.8 MHz for H, and 150.9 and 125.7 MHz for 13C,
respectively. Correlation spectroscopic 1H,1H-PFG-COSY and
1H,13C-HSQC experiments were applied for resolution and assign-
ment of resonance signals. ESI−MS and APCI−MS spectra were
measured with LC−MS LTQ-Orbitrap Velos (ThermoScientific,
Waltham, MA) and LC−MS TSQ Quantum Access Max Triple
Quadrupole mass spectrometers (Thermo Scientific, Waltham, MA).
Ultraviolet (UV) spectra were recorded using a Cary 50 UV−vis
spectrophotometer (Varian, Inc., Palo Alto, CA). Merck precoated
silica gel F254 plates were used for thin-layer chromatography (TLC).
Spots were detected by heating after spraying with 5% phosphomo-
lybdic acid in EtOH. Melting points were measured on a Boetius hot-
stage microscope and were not corrected.
Sunflower Bee Pollen Samples. Monofloral sunflower bee
pollen was obtained from the Slovak University of Agriculture in
Nitra, Slovak Republic (Assoc. Prof. Jan Brindza). The bee pollen
material was taken from the hive storages and stored in the freezer
until the time of chemical study. The wet bee pollen contained 15.5%
m/m of water, 14.0% m/m of proteins, 6.5% m/m of lipids, 17.0% of
carbohydrates, and 2.2% m/m of ash. Botanical origin of the raw
material was confirmed by scanning electron microscope (SEM)
analyses of the surface of bee pollen granules with a focused beam of
electrons (Figure S1).
+
C13H19N2O2 = 235.14410).
Feruloyl Putrescine Hydrochloride (1b). The product was
obtained in 89% yield as a yellowish solid. Mp = 113−115 °C; Rf =
0.58 (CH2Cl2/CH3OH/27% aq NH3, 2:2:1); UV (CH3OH) λmax 320
nm; 1H NMR (500 MHz, CD3OD) δ 1.66 (4H, m, H-3, 4), 2.93 (2H,
t, J = 7.3 Hz, H-2), 3.33 (2H, t, J = 6.6 Hz, H-5), 3.84 (3H, s, H-10′),
6.46 (1H, d, J = 15.7 Hz, H-8′), 6.76 (1H, d, J = 8.2 Hz, H-5′), 7.00
(1H, dd, J = 8.2 Hz, 2.0 Hz, H-6′), 7.10 (1H, d, J = 1.9 Hz, H-2′),
7.45 (1H, d, J = 15.7 Hz, H-7′); 13C NMR (126 MHz, CD3OD) δ
24.5 (C-4), 26.0 (C-3), 39.0 (C-2), 39.7 (C-5), 55.0 (C-10′), 110.2
(C-2′), 115.1 (C-5′), 122.3 (C-6′), 126.5 (C-1′), 127.0 (C-1′), 141.8
(C-7′), 148.0 (C-4′), 149.0 (C-4′), 168.4, 169.3, 170.4 (C-9′); IR
(KBr) ν = 3381, 1214 cm−1 (OH), 3020, 1168, 978 cm−1 (CH),
2932, 2856, 1444, 1363, 737 cm−1 (CH2), 1654, 1539, 1337 cm−1
(CONH), 1602, 1582, 1513 cm−1 (C = C), 1131, 830 cm−1 (CNC),
1108 cm−1 (OCH3), 1014 cm−1 (CC, CN), 515 cm−1 (skeletal);
Raman (λex 1064 nm) ν = 3064, 3019, 1168, 973 cm−1 (CH),
2983, 2937, 2867, 2857, 1442 cm−1 (CH2), 1733 cm−1 (CO),
1654, 1315 cm−1 (CONH), 1625, 1602, 1583, 1517 cm−1 (CC),
1222 cm−1 (OH), 1122, 828 cm−1 (CNC), 1081, 1029 cm−1 (CC,
CN), 644, 524, 449, 416, and 377 cm−1 (skeletal); HRESIMS m/z
Extraction and Fractionation of Crude Sunflower Bee
Pollen Extract. Monofloral sunflower bee pollen was pulverized.
Fine powder (12.684 g, moisture: 9.48%) was subjected to solvent
extraction with CHCl3/CH3OH mixture (2:1, v/v) at room
temperature for 6 h and filtered. The filtrate was evaporated under
reduced pressure to afford lipophilic extract (1.157 g), which was
purified by flash chromatography on silica gel (70−230 mesh) column
to yield three fractions: Fr-1 (eluted with petroleum ether/diethyl
+
265.15491 [M + H]+ (calcd for C14H21N2O3 = 265.15467).
B
DOI: 10.1021/acs.jafc.8b03976
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Figure 1. FT-IR spectra of fractions Fr-1−3.
N,N′-Di(p-coumaroyl) Putrescine (2). The product was
obtained as a pale yellow solid (560 mg). Rf = 0.84 (CH2Cl2/
CH3OH/27% aq NH3, 2:2:1); UV (CH3OH) λmax 308 nm; 1H NMR
(500 MHz, CD3OD) mixture of conformers, some hydrogen atoms
give more than one signal δ 1.66 (4H, m, H-3, 4); 3.36 (4H,
overlapped with solvent, H-2, 5); 6.43, 6.55, 6.60 (2H, d, J = 16.0 Hz,
H-8′); 6.80, 6.86, 7.22 (4H, d, J = 8.6 Hz, H-3′, 5′); 7.42, 7.56, 7.63
(4H, d, J = 8.6 Hz, H-2′, 6′); 7.47, 7.55, 7.83 (2H, d, J = 16.0 Hz, H-
7′); 13C NMR (126 MHz, CD3OD) mixture of conformers, some
carbon atoms give more than one signal δ 26.5, 26.6 (C-3, 4); 38.7,
38.8 (C-2, 5); 112.5, 117.0, 120.6 (C-8′); 115.3, 115.6, 122.0 (C-3′,
5′); 125.5, 126.3, 132.5 (C-1′); 128.5, 129.1, 130.2 (H-2′, 6′); 139.2,
140.3, 147.2 (C-7′); 152.1, 159.1, 160.5 (C-4′); 165.9, 167.1, 167.9
(C-9′); IR (KBr) ν = 3396 cm−1 (OH), 3022, 1172, 984 cm−1 (
CH), 2955, 2930, 2856, 1442, 1363 cm−1 (CH2), 3202, 1658, 1349
cm−1 (CONH), 1606, 1587, 1515 cm−1 (CC), 1134, 833 cm−1
(CNC), 1206, 1108 cm−1 (OCH3), 1014 cm−1 (CC, CN), 571, 520,
and 457 cm−1 (skeletal); Raman (λex 1064 nm) ν = 3388, 1207 cm−1
(OH), 3064, 3019, 1172, 979 cm−1 (CH), 2983, 2935, 1444, 1388,
1361 cm−1 (CH2), 1693 cm−1 (CO), 1656, 1317 cm−1 (CONH),
1633, 1604, 1587 cm−1 (CC), 1126, 863 cm−1 (CNC), 1023 cm−1
(CC, CN), 740, 644, 449, and 416 cm−1 (skeletal); APCI-MS m/z
solvent was evaporated under reduced pressure and residue was
worked up by column chromatography on silica gel to afford the
mixture (E)-4-O-acetylcoumaroyl amides of spermidine.15 Acetate
esters were deprotected by aqueous solution of NH3 (27%, v/v, 20
mL) in methanol (55 mL) under an inert atmosphere of argon. After
the mixture had been stirred for 5 h at 25 °C, the solvent was
evaporated under reduced pressure. The residue was taken up in
CH2Cl2 and evaporated to dryness to provide the mixture of di-p-
coumaroyl and tri-p-coumaroyl spermidines.16 Di- and tri-p-
coumaroyl spermidines were obtained in 18.5% yield.
Di- and Tri-p-coumaroyl Spermidines (3). The mixture of di-
and tri-p-coumaroyl spermidines (307 mg) was obtained as a
brownish solid. Rf = 0.76−0.85 (CH2Cl2/CH3OH/27% aq NH3,
2:2:1); UV (CH3OH) λmax 308 nm; 1H NMR (500 MHz, CD3OD) δ
1.59/1.75 (2H, m, H-8), δ 1.64/1.69 (2H, m, H-7), 1.87/1.96 (2H, q,
J = 7.0 Hz, H-3), 3.35/3.38 (4H, m, H-2, 9), 3.50/3.58 (4H, m, H-6),
3.54/3.60 (4H, m, H-4), 6.44 (d, J = 15.8 Hz, H-8′), 6.81 (d, J = 8.5
Hz, H-2′, 6′), 6.83/6.87 (d, J = 15.8 Hz, H-8′), 7.43 (d, J = 8.5 Hz, H-
3′, 5′), 7.47 (d, J = 15.8 Hz, H-7′), 7.52/7.59 (d, J = 15.8 Hz, H-7′);
13C NMR (126 MHz, CD3OD) δ 25.0/26.5 (C-7), 26.5/26.6 (C-8),
27.5/29.2 (C-3), 36.5/36.8 (C-2), 38.5/38.7 (C-9), 44.3/45.6 (C-4),
46.2/47.6 (C-6), 113.5/113.7 (C-8′), 115.4 (C-2′, 6′), 116.5 (C-8′),
126.2−126.5 (C-1′, 1′), 129.3 (C-3′, 5′), 141.5 (C-7′), 159.3 (C-4′),
167.8 (C-9′), 168.0 (C-9′); IR (KBr) ν = 3410, 1215 cm−1 (OH),
3006, 1277, 983 cm−1 (CH), 2958, 2871, 1450, 1350, 765 cm−1
(CH2), 3201, 1663, 1349 cm−1 (CONH), 1606, 1587, 1516 cm−1
(CC), 1127, 849 cm−1 (CNC), 1033 cm−1 (CC, CN), 569 and 457
cm−1 (skeletal); Raman (λex 1064 nm) ν = 3388, 1224 cm−1 (OH),
3072, 3004, 1162, 979 cm−1 (CH), 2983, 2935, 1452, 1430, 1388,
1363 cm−1 (CH2), 1697 cm−1 (CO), 1658, 1320 cm−1 (CONH),
1631, 1600, 1515 cm−1 (CC), 1128, 867 cm−1 (CNC), 1089, 1031
cm−1 (CC, CN), 651, 570, and 449 cm−1 (skeletal); APCI-MS m/z
584.27540 [M + H]+ and m/z 438.23864 [M + H]+ (calcd for
+
381.18117 [M + H]+ (calcd for C22H25N2O4 = 381.18088).
Synthesis of p-Coumaric Acid Amides of Spermidine. p-
Coumaric acid (1.09 g, 6.1 mmol) was acetylated in 15 mL of
tetrahydrofuran with 1.2 mL of acetyl chloride (16.9 mmol) in the
presence of pyridine (1.5 mL) for 12 h at 25 °C. The mixture was
worked up by column chromatography on silica gel (70−230 mesh)
to yield (E)-4-O-acetylcoumaric acid (5.44 mmol, 1.34 g, 89.3%). A
mixture of (E)-4-O-acetylcoumaric acid (5.44 mmol, 1.34 g) and
thionyl chloride (10 mL) was refluxed at 80 °C for 16 h. The mixture
was evaporated to dryness to afford (E)-4-O-acetylcoumaroyl chloride
and applied to the next step without further purification. To a stirred
solution of spermidine (1.64 mmol, 0.33 g) and triethylamine (5.5
mmol, 0.56 g) in anhydrous tetrahydrofuran (20 mL) on an ice water
bath was added dropwise a solution of (E)-4-O-acetylcoumaroyl
chloride. After the mixture had been stirred for 12 h at 25 °C, the
+
C25H32N3O4 = 438.23873).
Antifungal Activity Assay. The mold strain Aspergillus niger
CCM 8189 was obtained from Czech Collection of Microorganisms
(Brno, Czech Rep.); Fusarium culmorum DMF 0103 and Penicillium
C
DOI: 10.1021/acs.jafc.8b03976
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Journal of Agricultural and Food Chemistry
Article
Figure 2. FT-Raman spectra of fractions Fr-1−3.
verrucosum DMF 0023 were from the collection of the Department of
Dairy, Fat and Cosmetics (UCT Prague, Czech Rep.). Mold strains
were selected to test the growth inhibition activity of sunflower bee
pollen fractions, p-coumaroyl putrescine hydrochloride (1a), feruloyl
putrescine hydrochloride (1b), N,N′-di(p-coumaroyl) putrescine (2),
and p-coumaroyl spermidines (3). The molds were grown on malt
extract agar (MEA) plates at 25 °C for 72 h and maintained with
periodic subculturing at 4 °C. The spore suspension for further
inoculation was obtained by washing the particular plate with 5 mL of
physiological saline solution with Tween 80 (0.5%, v/v). The
concentration of spores was adjusted to 105 spores per mL by proper
dilution.17 Malt extract broth or agar was supplemented with various
concentrations (20, 10, 5, 2.5, and 1.25 mmol/dm3) of the tested
compounds and sterilized (121 °C, 15 min, 0.15 MPa) before
cultivation. Two different methods were used for antifungal activity
determination.
where S is the radial growth area (cm2) of molds on the plates with or
without phenolamides.
RESULTS AND DISCUSSION
■
Extraction and Fractionation of Crude Sunflower Bee
Pollen Extract. Specific secondary metabolites of pollen
grains were isolated, and the antifungal activity against
destructive and mycotoxigenic molds was assayed. The yield
of crude extract from sunflower bee pollen was 10.08% by dry
weight. TLC results confirmed the complex nature of extract
with dozens of potential antimicrobial compounds differing in
polarity. Bioassay-guided fractionation was achieved by flash
chromatography and resulted in three fractions. In the
separation profile, hydrophobic constituents of the fraction
Fr-1 (33.7%) were eluted quickly with petroleum ether/diethyl
ether from 100:0 to 87:13 followed by medium polar
compounds of the fraction Fr-2 (40.4%). A third ninhydrine
positive fraction (25.9%) was eluted with acetone and
methanol. Most analytes present in the obtained fractions
were in general weak chromophores. Therefore, identification
of structures was based on MS, NMR, and other spectroscopic
data and their comparison with literature. The FT-IR and FT-
Raman spectra of the fractions are represented in Figures 1 and
2, respectively.
First, growth curves were measured in microtitration plates by
spectrophotometric method using an automatic cultivator/reader
PowerWave XS (BioTek Instruments, Winooski, U.S.) according to
́
Kosova et al. (2015) with a slight modification: 2 μL of spore
suspension (105/mL) was inoculated into 200 μL of malt extract
broth enriched with different concentrations of tested compounds;
pure malt extract broth was used as the control. The optical density
was measured at 630 nm.17 All measurements were performed in
triplicate, and means were used for the calculation of minimal
inhibitory concentration (MIC) expressed as the lowest concentration
of assayed substances inhibiting the visible growth of molds.
Structural Elucidation of Fr-1. The FT-IR spectrum of Fr-1
(Figure 1, top) displayed two absorption bands at 1246 cm−1
(CO stretching) and 1739 cm−1 (CO stretching), which
were assigned to ester groups. The Raman band of esters was
found at 1729 cm−1 (CO stretching). The bands at 1667,
1631, and 1602 cm−1 arose from stretching vibrations of
unsaturated fatty acids and other unsaturated or aromatic
molecules or residues. The long alkyl H/C signals of saturated,
mono-, and polyunsaturated fatty acids predominates in the 1H
and 13C NMR spectra of Fr-1 (Table S1).20,21 The protons of
Second, the radial growth of molds was monitored on solid MEA
supplemented with 10, 5, 2.5, and 1.25 mmol/dm3 of p-coumaroyl
putrescine hydrochloride (1a) and feruloyl putrescine hydrochloride
(1b). Plates were inoculated by 5 μL of the spore suspension (105/
mL) in the middle of the dish and incubated at 25 °C for 10 days;
pure malt extract agar was used as the control.18,19 Antifungal
activities were calculated from the following equation:
i
y
Sinhibition
Sblank
j
z
j
z
inhibition = j1 −
z·100 [%]
j
j
z
z
(1)
k
{
D
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J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
glycerol moiety in TAG were found at δ 5.26 ppm (triplet of
triplets), 4.14, and 4.31 ppm (both are doublets of doublets).
The 13C NMR spectrum of Fr-1 showed signals of three enol
carbons of β-diketones at δ 194.65, 194.46, and 194.04 (Table
S2 and Figure S2).22 A broad proton signal at δ 15.50 arose
from enol hydroxyls. Several triplets at δ 2.26−2.49 and the
corresponding positive APT carbon signals at δ 38.36 and
43.73 were assigned to side CH2 attached to carbonyl in enol
form. The proton signals of phenyl ring conjugated with
ketone were found at δ 7.88 (ortho-position, doublet), δ 7.43
(meta-position, triplet), and δ 7.50 (para-position, triplet); the
corresponding negative APT carbon signals arose at δ 126.93,
δ 128.49, and δ 132.11, respectively. The H/C HMQC signals
at δ 2.42/38.99 and at δ 2.49/43.75, 43.69 were assigned to the
side CH2 attached to carbonyl in enol form. The H/C HMQC
signals of CH2/CH group between two carbonyls of
aliphatic β-diketones were found at δ 3.54/57.12 (ketone)
and at δ 5.47/98.96, 99.03 (enol); corresponding signals of
CH2/CH group between two carbonyls of aromatic β-
diketones were found at δ 4.08/53.92 (ketone) and at δ 6.17/
95.96 (enol).22
plane and out-of-plane vibrations of CH in phenolic acids.27
Highly overlapped bands in the region of 1000−1170 cm−1
arose from stretching vibrations of CO, CN, and CC bonds.
The band at 829 cm−1 was assigned to symmetric CNC
stretching in polyamines.28
The FT-Raman spectrum of the Fr-3 (Figure 2, bottom)
showed strong bands at 1641, 1606, and 1587 cm−1 and a
smaller one at 1517 cm−1 assigned to CC stretching
vibrations in phenolic acids.27 The bands at 1442, 1481, 2933,
and 2857 cm−1 were assigned to CH2 scissoring and stretching
vibrations in polyamines.28 The band at 1222 cm−1 was
assigned to OH in-plane bending in phenolics.29 Two bands at
3062 and 3019 cm−1 (CH stretching), 1172 cm−1 (CH
in-plane bending), and 975 cm−1 (CH out-of-plane
bending) arose from unsaturated and aromatic moieties.
Proton and 13C NMR spectra of Fr-3 showed intense
−CH signals of para-substituted phenyls found at δ 6.77
(ortho-position, doublet)/δ 116.0, δ 7.37/δ 130.7 (meta-
position, doublet) (Table S3). APT positive signals of
quaternary carbons were observed at δ 159.3 and δ 127.7.
The signals of conjugated CC bond were found at δ 6.38/δ
118.40 and δ 7.49/δ 141.93 and δ 7.52/δ 144.48. Finally,
several carbonyl carbon signals were found at δ 169.1−169.4
(amides), δ 170.3−170.6 (esters), and δ 171.8−172.0 (acids).
All of these signals confirmed the presence of para-substituted
phenols with conjugated double CC bond and carboxylic
group that is typical for p-coumaric acid and its conjugates
(esters, amides).29 Several methylene proton signals at δ 1.55−
1.95 (internal), δ 3.09−3.38 (bound to end amine nitrogen),
and δ 3.18−3.58 (bound to internal amine nitrogen) and the
corresponding carbon signals at δ 25.5−29.8, 37.8−38.3, and δ
39.7−47.7 confirmed the presence of spermidine molecules
conjugated with phenolic acids.30 Thus, the main compound
found in the methanol/ethanol soluble fractions was tri-p-
coumaroylspermidine31 accompanied by other HCAA of
spermidine and putrescine. LC−MS analyses confirmed exact
masses of 584.27540 [M + H]+ and m/z 438.23864 [M + H]+
corresponding to tri- and di-p-coumaroylspermidines. These
compounds have been previously found in sunflower pollen,
and their phagostimulatory activities were described. Further
acetone fractionation removed insoluble glyceroglycolipids
representing 0.54 g/100 g of dry matter of bee pollen grains.
Residue of Fr-3 (2.07 g/100 g) was identified as the mixture of
predominating hydroxycinnamic acid amides accompanied by
glycerophospholipids based on LC−MS measurements. The
results of fractionation were in accordance with literature
data.32 Less intense signals of other phenolic acids were also
found. The HMQC signal at δ 3.64/δ 51.99 arose from
methoxy groups in ferulic acids. Structural elucidation of Fr-3
resulted in the synthesis of basic and neutral HCAA
derivatives.
Structural Elucidation of Fr-2. The FT-IR spectrum of the
Fr-2 (Figure 1, middle) displayed strong absorption bands of
CO stretching vibrations at 1712 and 1737 cm−1 that arose
from carboxylic and ester groups, respectively. The Raman
bands at 1729 and 1716 cm−1 were assigned to CO
stretching in esters and acids, respectively. The bands at 1656
and 1602 cm−1 originated from CC stretching vibrations in
1
unsaturated residues. Like in the case of Fr-1, the H and 13C
NMR spectra of Fr-2 showed a lot of characteristic signals
originated from monoacyglycerols, free fatty acids, and fatty
alcohols (Table S1).20,21
In addition, the signals of sterols, terpenes, and many other
cyclic compounds were also found.23−25 Allyl moiety
characteristic for ent-kaurenes (diterpenoids) was found at δ
155.5, 155.7, and 156.7.23 Carbon signals at δ 64.3, 65.4, 77.2,
and 79.0 arose from CH2OH and CHOH of hydroxylated ent-
kaurenes; carbonyl carbons at δ 183.7−183.8 indicated the
carboxylic group attached at C-4.24,26 These compounds
showed inhibitory effects on activation Epstein−Barr virus.
Isolated fractions Fr-1, Fr-2, however, did not show
prospective antifungal effects comparable to crude extract
and Fr-3.
Structural Elucidation of Fr-3. Appreciable antifungal
activities against A. niger CCM 8189, F. culmorum DMF
0103, and P. verrucosum DMF 0023 were observed only in the
case of Fr-3. Minimum inhibition concentrations of ninhydrine
positive Fr-3 on all of the tested fungi ranged from 5 to 10 g/
dm3 obtained by microtitration screening method. Crude
nonfractionated extract showed a prospective antifungal effect
at higher concentrations (>20 g/dm3). The FT-IR spectrum of
the Fr-3 (Figure 1, bottom) displayed strong absorption bands
of aromatics and sugars, while the bands of alkyls are relatively
weak. A strong broad band with a maximum at 3397 cm−1
arose from OH vibrations in water, sugars, and phenolic
compounds. Bands at 1607 and 1513 cm−1 were assigned to
CC stretching vibrations of phenolic acids and their
derivatives (amides, esters).27 Bands at 1649, 1559, and 1542
cm−1 indicated the presence of amide groups. Two bands at
1454 and 1437 cm−1 were assigned to CH2 scissoring
vibrations in polyamines.28 The band at 1378 cm−1 is
characteristic for symmetric bending vibration of CH3 in
ferulic acid. Bands at 1275 and 981 cm−1 were assigned to in-
Synthesis of p-Coumaric and Ferulic Acid Amides of
Putrescine and Spermidine. A number of scientific
publications are devoted to the antimicrobial effects of plant
extracts containing HCAA, whether on the growth of
bacteria1,33 or fungi.34 Most authors, however, deal only with
the effect of the crude extracts containing dozens of potential
antimicrobial compounds, which may exhibit synergistic
antifungal activities as proved for lactic and acetic acids.35
Therefore, it is important to study pure compounds first to
accurately determine their effect.
Hydroxycinnamic acid amides of putrescine and spermidine
were difficult to isolate from natural sources such as sunflower
E
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Journal of Agricultural and Food Chemistry
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bee pollen in sufficient yield and required purity. Thus, p-
coumaroyl putrescine hydrochloride (1a), feruloyl putrescine
hydrochloride (1b), N,N′-di(p-coumaroyl) putrescine (2), and
p-coumaroyl spermidines (3) were synthesized (Figure 3 and
4) in good to quantitative yield.
Figure 3. Synthesis of p-coumaroyl putrescine hydrochloride (1a) and
feruloyl putrescine hydrochloride (1b).
Figure 5. 1H,13C HMBC signals assigned to interactions between
methylene protons and carbonyl carbons that confirmed amide
linkages in p-coumaroyl spermidines and putrescines.
putrescine hydrochloride (1a), feruloyl putrescine hydro-
chloride (1b), neutral N,N′-di(p-coumaroyl) putrescine (2),
and the mixture of p-coumaroyl spermidines (3) on the growth
inhibition of representative bee pollen contaminants A. niger
CCM 8189, F. culmorum DMF 0103, and P. verrucosum DMF
0023.5 Moreover, Aspergillus spp., Fusarium spp., and
Penicillium spp. are mycotoxigenic fungi producing aflatoxins,
ochratoxins, trichothecenes, and many other mycotoxins under
optimal growth conditions.5,7 The recovery of these molds
represents a potential risk for human health.
Figure 4. Synthesis of di- and tri-p-coumaroyl spermidines (3).
FTIR, FT-Raman, 1H, and 13C APT NMR spectra of
synthesized phenolamides 1a, 1b, 2, and 3 confirmed the
presence of structural parts of the corresponding phenolic acids
and polyamines, that is, phenylpropenoyl and N-methylene
groups. For example, IR and/or Raman bands at 1625−1633,
1601−1606, 1580−1587, 1513−1517, 1162−1175, and 978−
984 cm−1 (CC stretching, CH in-plane and out-of-plane
bending) are characteristic of phenolic acids.27 The IR and/or
Raman bands of polyamines were found at 2983, 2958−2929,
2851−2871, 1430−1452, 1347−1388, and 736−765 cm−1
(vibrations of CH2).28 The vibration bands characteristic for
amide bonds were found at 3202−3201, 1643−1663, 1537−
1539, and 1315−1349 cm−1 (vibrations amide A, I, II, and III,
respectively) that confirm amidation. Proton and 13C NMR
spectra of the products were very complicated because of the
presence of several conformers of the polyamine chain.
Nevertheless, all of the proton and carbon signals of
phenolamides were assigned by using correlation NMR
experiments COSY, HMQC, and HMBC. The APT signals
of amide carbonyl carbons were found at δ 167.2−168.0
(secondary and tertiary amides) and δ 168.3 (primary amide).
First, the growth of tested molds in microtitrate plates was
used as a screening method to determine the range of required
substance concentrations. As can be seen from Table 1,
Table 1. Antifungal Activity of Hydroxycinnamic Acid
Amides on the Growth Inhibition of Aspergillus niger,
Fusarium culmorum, and Penicillium verrucosum Determined
on the Microtitration Plates
F. culmorum,
P. verrucosum,
A. niger,
a
tested compound/MIC
mmol/dm3
mmol/dm3
mmol/dm3
p-coumaroyl putrescine
hydrochloride (1a)
5
2.5
10
feruloyl putrescine
hydrochloride (1b)
5
2.5
20
N,N′-di(p-coumaroyl)
putrescine (2)
di- and tri-p-coumaroyl
spermidines (3)
20
20
>20
5
>20
5
a
MIC showed the minimal inhibitory concentration expressed as the
lowest concentration of assayed substances inhibiting the visible
growth of molds.
1
The H,13C HMBC signals at δ 3.35/167.9, 3.37/168.0, 3.56/
167.8, 3.58/167.7, 3.49/167.5, and 3.50/167.5 confirmed the
connection between carbonyl carbon of phenolic acids and
methylene hydrogens of polyamine residues via amide bonds
(Figure 5).
Antifungal Activity of Hydroxycinnamic Acid Amides.
Sunflower bee collected pollen is now used as a supplement in
the human diet, and therefore the assessment of its
microbiological quality is very important. The aim of the
antifungal test was to verify if the compounds naturally
occurring in bee pollen can reduce the growth of commonly
widespread molds. Here, we present the effect of p-coumaroyl
selected phenolic compounds showed significantly different
inhibitory effects on the growth and survival of tested molds. A.
niger CCM 8189 (5−20 mmol/dm3) and F. culmorum DMF
0103 (5−20 mmol/dm3) species exhibited higher resistance to
the antifungal effects of hydroxycinnamic acid amides, whereas
P. verrucosum DMF 0023 was the most sensitive during the
measurement on the microtitration plate (Table 1). Di- and
tri-p-coumaroyl spermidines (3) showed a MIC of 5 mmol/
dm3 for P. verrucosum and A. niger. Minimum inhibition
F
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Journal of Agricultural and Food Chemistry
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Figure 6. Mycelium growth inhibition of tested molds on malt extract agar with different concentrations (0−10 mmol/dm3) of p-coumaroyl
putrescine hydrochloride (1a) and feruloyl putrescine hydrochloride (1b) after 10 days at 25 °C. The mycelium growth inhibition was calculated
through eq 1 (see Material and Methods).
concentrations of p-coumaroyl putrescine hydrochloride (1a)
on all of the tested mycotoxigenic fungi ranged from 2.5 to 10
mmol/dm3 (Table 1). Lipophilic character of putrescine
derivatives was considered as a key factor in determining
solubility and antifungal activity. HCAA in the form hydro-
chloride had the highest inhibitory effects and the lowest
lipophilicity based on partition coefficient (Ko/w). Positively
charged compounds (1a, 1b) with protonated amino groups at
physiological pH could play a defensive role against fungal
spoilage by electrostatic interactions with negatively charged
proteins, phospholipids, and nucleic acids, while neutral HCAA
could serve as antioxidant and the pool of antifungal agents.36
On the basis of the previous results, p-coumaroyl putrescine
hydrochloride (1a) and feruloyl putrescine hydrochloride (1b)
were used for the determination of fungal radial growth
inhibition on a solid media. The results (Figure 6) showed that
the MIC values detected for the most sensitive P. verrucosum
DMF 0023 by spectrophotometric method (2.5 mmol/dm3)
differed from the growth inhibition on agar media (10 mmol/
dm3). The method used may influence the determination of
the MIC. The minimum inhibitory concentrations of tested
substances 1a and 1b found by microplate cultivation were
lower than those on solid agar culture. Concentrations of these
substances of 2.5 mmol/dm−3 for F. culmorum and 5 mmol/
dm−3 for P. verrucosum prevented mold growth completely
(Table 1). In the case of growth on malt extract agar, only 40%
inhibition of growth was observed at these concentrations.
Differences between these methods may be due to different
growth rates during growth in liquid and solid media. Solid
culture media usually allow regular growth and allow the mold
to sporulate more easily as compared to liquid media. On the
basis of practical experience, the cultivation on solid media can
be rather recommended, also due to better access of mold to
oxygen.37
Antifungal activity of p-coumaroyl putrescine hydrochloride
(1a) was slightly increased in relation to that of feruloyl
putrescine hydrochloride (1b). p-Coumaroyl putrescine hydro-
chloride could be considered an analogue of benzoic acid and
G
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Journal of Agricultural and Food Chemistry
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its derivatives, fundamental industrial biocide. The presence of
the methoxy group in feruloyl putrescine hydrochloride
increased a bit the compound polarity and decreased the
biocide action on lipophilic outer membrane of molds (Table
1). Tested molds could be classified according to the inhibitory
effect of positively charged HCAA on the radial growth in
ascending order: A. niger CCM 8189 < Fusarium culmorum
DMF 0103 < Penicillium verrucosum DMF 0023.
As can be seen from Figure 6, the addition of assayed
compounds did not affect only the radial growth of molds, but
also their ability to sporulate. The spore formation of Fusarium
culmorum was significantly slowed. Microscopically, the
occurrence of macroconidia in the randomly selected 15 visual
fields of the control and supplemented samples by HCAA in
the culture medium was controlled. The number of F.
culmorum macroconidia was noticeably reduced during the
growth on agar with the addition of tested substances (data not
shown). Moreover, results demonstrated that positively
charged HCAA affected colony morphology.38 Colonies of
Aspergillus spp., Fusarium spp., and Penicillium spp. species with
mycelium growth inhibition above 30% were not pigmented or
adopted lighter coloration in comparison with control
experiments.
PUFA, polyunsaturated fatty acid; TAG, triacyglycerol; EI−
MS, electron impact mass spectrometry; TIC, total ion current
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* Supporting Information
The Supporting Information is available free of charge on the
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AUTHOR INFORMATION
Corresponding Author
*E-mail: jan.kyselka@vscht.cz.
ORCID
Jan Kyselka: 0000-0003-4071-0480
Funding
This work was supported from specific university research
(MSMT no. 20-SVV/2017).
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HCAA, hydroxycinnamic acid amides; TMV, tobacco mosaic
virus; FTIR, Fourier-transform infrared spectroscopy; FT,
Fourier transformation; NMR, nuclear magnetic resonance;
HSQC, heteronuclear single-quantum coherence; COSY,
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TLC, thin-layer chromatography; SEM, scanning electron
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hexylcarbodiimide; CCM, Czech Collection of Microorgan-
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DOI: 10.1021/acs.jafc.8b03976
J. Agric. Food Chem. XXXX, XXX, XXX−XXX