Microbial transformations of 4′-methylchalcones as an efficient method of obtaining novel alcohol and dihydrochalcone derivatives with antimicrobial activity

Article abstract of DOI:10.1039/c8ra04669g

Biotransformations are an alternative method of receiving dihydrochalcones as a result of the reduction of α,β-unsaturated ketones-chalcones. In presented research, two strains of bacteria-Gordonia sp. DSM44456 and Rhodococcus sp. DSM364-were selected as

Full text of DOI:10.1039/c8ra04669g

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Microbial transformations of 40-methylchalcones as
an efficient method of obtaining novel alcohol and
dihydrochalcone derivatives with antimicrobial
activity†
Cite this: RSC Adv., 2018, 8, 30379
a
a
Barbara Z˙arowskab
*
Joanna Kozłowska,
and Mirosław Anioła
Bartłomiej Potaniec,
Biotransformations are an alternative method of receiving dihydrochalcones as a result of the reduction of
a,b-unsaturated ketones – chalcones. In presented research, two strains of bacteria – Gordonia sp.
DSM44456 and Rhodococcus sp. DSM364 – were selected as effective biocatalysts that are able to
transform chalcones in a short period of time. As a result of our investigation 3 new dihydrochalcones
and one novel alcohol were obtained with high isolated yields. All 40-methylchalcone derivatives and
biotransformations products were tested for antimicrobial activity against Escherichia coli ATCC10536,
Staphylococcus aureus DSM799, Candida albicans DSM1386, Alternaria alternata CBS1526, Fusarium linii
KB-F1, and Aspergillus niger DSM1957. The best inhibitory effect was observed for all chalcones against
E. coli ATCC10536 – compounds 1–6 and 8 prevented thorough growth of this strain (DOD ¼ 0).
Moreover, dihydrochalcones showed about 2–3 times stronger inhibitory effect against S. aureus
DSM799 in comparison to their chalcones. Excluding the E. coli ATCC10536 strain, 3-(4-carboxyphenyl)-
1-(4-methylphenyl)propan-1-ol (8b) had weaker biological activity than 4-carboxy-40-methyl-a,b-
dihydrochalcone (8a).
Received 31st May 2018
Accepted 22nd August 2018
DOI: 10.1039/c8ra04669g
rsc.li/rsc-advances
potential anticancer agents have already been found among
chalcones. For example, 20,60-dihydroxy-404-dimethoxydihy-
drochalcone, 20,60-dihydroxy-40-methoxydihydrochalcone and
Introduction
Chalcones (1,3-diaryl-2-propen-1-ones) belong to a wide group
of polyphenols known as avonoids. Their chemical structure is
characterized by the a,b-unsaturated bond formed as the result
of opening ring C in avanones.1 The presence of numerous
electron-withdrawing or donating groups allows to describe
chalcones as compounds with various biological activities such
as anti-inammatory,2 antioxidant,3 antibacterial4 and
antitumor.5,6
20,40,60-trihydroxydihydrochalcone (phloretin) augmented
apoptosis in prostate cancer cells.9
According to literature, the chemoselective reduction of the
a,b-unsaturated bond is the result of hazardous reagents – some
of them requiring transition metals for reaction.10,11 Microbial
transformations are a good alternative to chemical synthesis,
transforming chalcones into dihydrochalcones without using
harmful and toxic compounds. Also, the application of whole
cells of microorganisms is oen superior to enzymatic modi-
cations, that usually are more costly, and need additional
cofactors and strict incubation conditions. Moreover, multistep
reactions, impeded by the traditional approach of chemical
synthesis, are possible with the enzymatic apparatus of
microbials.
The most frequently described modications of avonoids
catalysed by microbial cells are hydroxylation, dehydroxylation,
O-methylation, O-demethylation, glycosylation, deglycosylation,
dehydrogenation, hydrogenation, C ring cleavage, cyclization
and reduction.12 Enoate reductase has been characterised as
one of the main enzymes responsible for reduction carbon–
carbon double bonds. It belongs to the Old Yellow Enzyme
Hydrogenation of the a,b-unsaturated bond in chalcones
leads to derivatives called dihydrochalcones. Interestingly,
neohesperidin occurring in citrus fruits is characterized with
a
bitter taste in contrast to the neohesperidin dihy-
drochalcone – hypocaloric sweetener accepted by the EU and
FDA as a food additive.7 Furthermore, antioxidant properties
of dihydrochalcones were observed to be signicantly
stronger than corresponding avanones.8 Moreover, many
aDepartment of Chemistry, Wrocław University of Environmental and Life Sciences,
Norwida 25, 50-375 Wrocław, Poland. E-mail: joannakozlowska3@gmail.com
bDepartment of Biotechnology and Food Microbiology, Wrocław University of
´
Environmental and Life Sciences, Chełmonskiego 37, 51-630 Wrocław, Poland
† Electronic supplementary information (ESI) available: 1H, 13C NMR of all
products and FTIR-ATR of compounds 4a, 5a, 8a and 8b. See DOI:
10.1039/c8ra04669g
(OYE)
family,
consisting
of
the
avin-dependent
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oxidoreductases, that require NADH to be active.13 Current 10 g peptone, 2 g casein hydrolysate, 2 g yeast extract, 6 g
knowledge proofs the presence of enoate reductases in known sodium chloride and 20 g glucose dissolved in 1000 mL distilled
biocatalysts such as bacteria and fungi strains.14,15 Additionally, water. Sterile culture medium was inoculated using 0.1 mL of
recent a report described the bioconversion of trans-chalcone by bacteria suspension and kept at 28 ꢀC on rotary shaker. Aer 48
whole cells of cyanobacteria.16 This variety of active whole-cell hours, 1 mg of 40-methylchalcone derivative dissolved in 0.5 mL
catalysts creates a possibility of obtaining novel derivatives acetone (1–7) or dimethyl sulfoxide (DMSO) (8) was added to the
with enhanced biological properties. In recent years, antimi- culture. Progress of biotransformation was monitored by the
crobial activity of avonoids has gained much interest.
thin layer chromatography (TLC) and the liquid high-
Infections caused by microorganisms are one of the most performance chromatography (HPLC).
important problems in all aspects of our life, especially in
schools, hospitals and workplaces. Increasing resistance of
microorganisms to commonly used antibiotics encourages the
Biotransformation in preparative scale
exploration of novel active compounds, oen from natural To isolate products of biotransformation observed in small
sources. The variety of chalcones and dihydrochalcones deriv- scale, preparative investigation was performed using 2000 mL
atives can hinder bacteria growth, including multi-resistant Erlenmeyer asks containing 300 mL of sterile culture medium,
strains.4,17 Phloretin and their glycosylated derivatives – phlor- inoculated with 1 mL of bacteria suspension and maintained at
izin and phloretin 30,50-di-C-glucoside – are known growth 28 ꢀC on rotary shaker. Aer 48 hours 30 mg of 40-methyl-
inhibitors of Gram-positive bacteria such as Staphylococcus chalcone derivative dissolved in 1 mL acetone (1–7) or DMSO (8)
aureus ATCC6538, clinical strains of methicillin-resistant was added to the grown culture and incubation under the same
Staphylococcus aureus (MRSA) and Listeria monocytogenes conditions was continued. Aer determining the complete
ATCC13932. Furthermore, phloretin showed antibacterial substrate conversion, biotransformation mixture was extracted
activity against Gram-negative bacteria – Salmonella typhimu- with ethyl acetate (3 ꢁ 100 mL). Organic fractions were collected
rium ATCC13311.18 Also, the investigation performed by and dried over anhydrous sodium sulphate. Organic solvent was
a scientic group in Brazil showed that 4-methoxy-40-methyl- evaporated on vacuum evaporator and biotransformation
chalcone (3) hindered the growth of Gram-positive bacteria extract was puried on column chromatography. Purity of ob-
Mycobacterium tuberculosis H37Rv with a MIC50 value of 66.9 tained products was veried by HPLC and structures were
1
mM.19
determined using H and 13C NMR.
In our paper, we describe microbial transformation of series
of 40-methylchalcone derivatives (1–8) performed by two species
of aerobic bacteria – Gordonia sp. DSM44456 and Rhodococcus
Analytical methods
sp. DSM364. Previous experiments carried out by our research Progress of biotransformations performed in small and
team allowed us to select two strains of bacteria capable of preparative scale was analysed by thin layer chromatography
transforming chalcones to dihydrochalcones with high isolated (TLC) on silica gel-coated aluminium plates with uorescent
yields.14 Moreover, we observed that elongation of biotransfor- indicator (DC-Alufolien, Kieselgel 60 F254; Merck, Darmstadt,
mation time provides a second metabolite: the alcohol. Subse- Germany) using mixture of hexane : ethyl acetate or chlor-
quently, an evaluation of antimicrobial activity against six oform : methanol as eluents. Products were detected by spray-
strains of microorganisms Escherichia coli ATCC10536, Staphy- ing the plates with a solution of 1% Ce(SO4)2 and 2%
lococcus aureus DSM799, Candida albicans DSM1386, Alternaria H3[P(Mo3O10)4] in 5% H2SO4 and subsequently visualised by
alternata CBS1526, Fusarium linii KB-F1, and Aspergillus niger heating. Extracts obtained from preparative biotransformations
DSM1957 was prepared for synthetized chalcones (1–8) and were puried by liquid column chromatography using silica gel
compared with their biotransformation products (1a–8a, 8b).
(Kieselgel 60, 230–400 mesh, Merck). The purity of the products
was analysed by HPLC on a Waters 2690 (Milford, MA, USA) with
Photodiode Array Detector Waters 996. The samples of
substrates and biotransformation products for HPLC analysis
were dissolved in methanol. The HPLC apparatus was equipped
with a reverse-phase C-18 column (Phenomenex, Torrance, CA,
United States, Kinetex 5u XB-C18 100A, 250 mm ꢁ 4.6 mm),
Experimental
Materials
All 40-methylchalcone derivatives (1–8) were prepared with the
procedure described by Amir et al. with yield 60–94%.20
Biotransformations were performed using two strains of
ꢀ
which was thermostated at 28 C, and analysed samples were
kept at 12 ꢀC. The mobile phase consisted of two eluents: A—1%
HCOOH in MeCN and B—1% HCOOH in H2O. Elution gradient
was started from 55% of eluent A to 45% of eluent B over
15 min. A ow rate of 1.5 mL minꢂ1 was used.
The structure of the obtained compounds was conrmed
using nuclear magnetic resonance (NMR) analysis. 1H-NMR and
13C-NMR spectra were recorded on a Bruker Avance™600 MHz
bacteria
– Gordonia sp. DSM44456 and Rhodococcus sp.
DSM364. Strains were obtained from the German Collection of
Microorganisms and Cell Cultures GmbH (DSMZ, Deutsche
Sammlung von Mikroorganismen und Zellkulturen, GmbH).
Biotransformation in small scale
Screening procedure was performed in Erlenmeyer asks (100 spectrometer (Bruker, Billerica, MA, USA) with chloroform-d as
mL) containing 25 mL of culture medium, which consisted of solvent.
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Positive and negative-ion HR ESI-MS spectra were measured for each strain were prepared as a mean values of the measured
on a Bruker ESI-Q-TOF Maxis Impact Mass Spectrometer OD as a function of time. The resulting antimicrobial activity
(Bruker, Billerica, MA, USA). The direct infusion of ESI-MS was expressed as the increase in optical density (DOD) in
parameters: the mass spectrometer was operated in positive comparison to the control cultures in the medium supple-
(1a–8a) and negative (8b) ion mode with the potential of 3.5 kV mented with dimethyl sulfoxide.
between the spray needle and the orice, nebulizer pressure of
0.4 bar, and a drying gas ow rate of 3.0 L minꢂ1 at 200 ꢀC. The
Results and discussion
Biotransformations
sample ow rate was set as 3.0 mL minꢂ1. Ionization mass
spectra were collected at the ranges m/z 50–1250.
Infrared spectra were determined using a Nicolet 6700 FTIR
spectrometer (Thermo Scientic, Waltham, MA, USA) with ATR
accessory with diamond crystal in the wavelength range 400–
Chalcones received in classical Claisen-Schmidt reaction were
used as a substrate to biotransformation.
Current literature describes the biotransformation of 40-
methylchalcone (1) to dihydrochalcone (1a) in Corynebacte-
rium equi IFO 3730 culture aer 3 days with 94% yield.21
However, our investigation have showed complete conversion
of all substrates (1–8) already aer maximum 24 hours with
isolated yields up to 99%. Microbial transformations were
monitored in time using thin layer chromatography and high-
performance liquid chromatography. Aer the disappearance
of the substrate, extraction and purication on liquid chro-
4000 cmꢂ1
.
Optical rotation was measured on Jasco P-2000 automatic
´
polarimeter (ABL&E-JASCO, Krakow, Poland). Solution was
prepared in chloroform and the concentration was expressed in
g/100 mL.
Biological assays
To evaluate activity of 40-methylchalcones and their derivatives matography gave us pure product. The structure of all the
obtained as a result of biotransformations, a series of antimi- compounds was determined by nuclear magnetic resonance
crobial assays were performed. In our investigation two strains analysis. Biotransformations of substrates 1–7 resulted in
of bacteria: E. coli ATCC10536 and S. aureus DSM799 and four obtaining their dihydrochalcones (Scheme 1). Interestingly,
strains of fungi: C. albicans DSM1386, F. linii KB-F1, A. alternata the product of 4-carboxy-40-methylchalcone (8) transformation
CBS1526 and A. niger DSM1957 were used. All the microorgan- under the same cultivation condition appeared to be the
isms were obtained from the collection of the Faculty of alcohol (8b), not the expected dihydrochalcone. The optical
Biotechnology and Food Microbiology, Wroclaw University of rotation of novel 3-(4-carboxyphenyl)-1-(4-methylphenyl)
Environmental and Life Sciences. Bacteria were cultured in the propan-1-ol (8b) was measured in chloroform and the value
standard nutrient broth (Biocorp, Warsaw, Poland), and fungi [a]2D0 ¼ ꢂ2.733 (c ¼ 0.9) was obtained. This result proved, that
in the YM medium, which consisted of 3 g yeast extract, 3 g malt product of biotransformation was a mixture of isomers with
extract, 5 g bacteriological peptone and 10 g of glucose dissolved a predominance of one of them. The rapidity of reduction led
in 1 L of distilled water. Assays were performed on 100-well us to analyse the biotransformation process in shorter inter-
microtiter plates, with the working volume of 300 mL per well: vals of time (aer 1, 3, 6, 9, 12 and 24 hours). The results are
280 mL of culture medium, 10 mL of microorganism suspension shown in Scheme 2. Aer 1 hour of biotransformation of 4-
and 10 mL of 40-methylchalcone or its derivative dissolved in carboxy-40-methylchalcone (8), the complete conversion to
DMSO (0.3% (w/v)). The nal concentration of the tested compound 8a by both strains was observed. Furthermore, aer
substance was 0.1% (w/v). Each culture was prepared in tripli- 6 hours of microbial transformation the second product (8b)
cate. The optical density (OD) of the cell suspensions was was observed. In addition, the conversion in Rhodococcus sp.
measured on Bioscreen C (Automated Growth Curve Analysis DSM364 culture was faster in comparison to Gordonia sp.
System Lab System, Helsinki, Finland) at 560 nm automatically, DSM44456. Both products of the biotransformation of 4-car-
at regular intervals of 30 min for 2–3 days. Cell cultures were boxy-40-methylchalcone (8) were isolated. The NMR analysis
maintained at 28 ꢀC on a continuous shaker. The growth curves conrmed our supposition that the product observed aer 1
Scheme 1 Biotransformations of 40-methylchalcone derivatives in Gordonia sp. DSM44456 and Rhodococcus sp. DSM364 cultures.
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Scheme 2 The content of biotransformation products (8a, 8b) of 4-carboxy-40-methylchalcone (8) in Gordonia sp. DSM44456 (A) and Rho-
dococcus sp. DSM364 (B) cultures (according to HPLC).
d 199.00 (C]O), 143.94 (C-40), 141.52 (C-1), 134.51 (C-10), 129.39
Table 1 Isolated yields (%) of biotransformation products in Gordonia
(C-20, C-60), 128.63 (C-3, C-5), 128.54 (C-2, C-6), 128.28 (C-30, C-
sp. DSM44456 and Rhodococcus sp. DSM364 culture (compounds
1a–7a and 8b isolated after 24 h, compound 8a isolated after 1 h)
50), 126.21 (C-4), 40.47 (C-a), 30.34 (C-b), 21.76 (C-40-CH3); HR
ESI-MS m/z calculated for C16H17O [M + H]+ 225.1274, found [M
Product
Gordonia sp.
Rhodococcus sp.
+ H]+ 225.1282.
40-Methyl-4-methyl-a,b-dihydrochalcone (2a), white solid,
mp 58–60 ꢀC (lit.25 59–61 ꢀC); 1H NMR (600 MHz, CDCl3): d 7.88–
7.84 (2H, m, H-20, H-60, AA0BB0), 7.25 (2H, d, J ¼ 8.7 Hz, H-30, H-
50), 7.15 (2H, d, J ¼ 7.9 Hz, H-3, H-5), 7.12 (2H, d, J ¼ 7.9 Hz, H-2,
H-6), 3.29–3.22 (2H, m, H-a), 3.06–2.99 (2H, m, H-b), 2.41 (3H, s,
C-40-CH3), 2.33 (3H, s, C-4-CH3); 13C NMR (150 MHz, CDCl3):
d 199.14 (C]O), 143.91 (C-40), 138.42 (C-4), 135.70 (C-1), 134.54
(C-10), 129.39 (C-3, C-5), 129.31 (C-30, C-50), 128.42 (C-2, C-6),
128.29 (C-20, C-60), 40.65 (C-a), 29.94 (C-b), 21.77 (C-40-CH3),
21.14 (C-4-CH3); HR ESI-MS m/z calculated for C17H19O [M + H]+
239.1430, found [M + H]+ 239.1435.
4-Methoxy-40-methyl-a,b-dihydrochalcone (3a), white solid,
mp 54–55 ꢀC (lit.26 57–58 ꢀC); 1H NMR (600 MHz, CDCl3): d 7.89–
7.82 (2H, m, H-20, H-60, AA0BB0), 7.29–7.19 (2H, m, H-30, H-50,
AA0BB0), 7.21–7.12 (2H, m, H-2, H-6, AA0BB0), 6.87–6.81 (2H, m,
H-3, H-5, AA0BB0), 3.79 (3H, s, C-4-OCH3), 3.28–3.20 (2H, m, H-
a), 3.04–2.96 (2H, m, H-b), 2.41 (3H, s, C-40-CH3); 13C NMR (150
MHz, CDCl3): d 199.17 (C]O), 158.08 (C-4), 143.90 (C-40), 134.56
(C-10), 133.54 (C-1), 129.46 (C-2, C-6), 129.39 (C-20, C-60), 128.29
(C-30, C-50), 114.04 (C-3, C-5), 55.40 (C-4-OCH3), 40.73 (C-a),
29.50 (C-b), 21.75 (C-40-CH3); HR ESI-MS m/z calculated for
1a
2a
3a
4a
5a
6a
7a
8a
8b
62.9
55.0
84.1
80.2
37.7
43.3
79.0
86.0
71.9
77.5
50.7
82.6
96.4
40.3
40.6
70.9
99.1
82.0
hour was 4-carboxy-40-methyl-a,b-dihydrochalcone (8a). The
presence of electron withdrawing group (e.g. carboxylic, nitro
group) usually enhance the rate of hydrogenation catalyzed by
enoate reductase.22 However, accelerated process of biotrans-
formation was observed only in the case of 4-carboxy-40-
methylchalcone (8). Most likely, the effect of action was con-
nected with better solubility of substrate or uptake by micro-
organism cells caused by presence of carboxylic group.23
The efficiency of microbial transformation in Gordonia sp.
DSM44456 and Rhodococcus sp. DSM364 culture described
Table 1.
C
17H19O2 [M + H]+ 255.1380, found [M + H]+ 255.1381.
2,4-Dimethoxy-40-methyl-a,b-dihydrochalcone (4a), yellow
Spectral data of biotransformation products
oil; FTIR-ATR (cmꢂ1): 2930.60, 2835.33, 1678.26, 1607.03,
1587.71, 1506.01, 1456.13, 1290.57, 1205.54, 1179.77, 1150.03,
The structures of biotransformation products were determined
by 1H-NMR, 13C-NMR and HR ESI-MS analysis. Additionally,
FTIR-ATR spectra of novel compounds were recorded. All data
are described in details below.
40-Methyl-a,b-dihydrochalcone (1a), white solid, mp 61–63 ꢀC
(lit.24 61–63 ꢀC); 1H NMR (600 MHz, CDCl3): d 7.90–7.83 (2H, m,
H-20, H-60, AA0BB0), 7.33–7.24 (6H, m, H-2, H-3, H-5, H-6, H-30, H-
50), 7.24–7.19 (1H, m, H-4), 3.32–3.25 (2H, m, H-a), 3.11–3.02
(2H, m, H-b), 2.41 (3H, s, C-40-CH3); 13C NMR (150 MHz, CDCl3):
1
1035.53, 824.73; H NMR (600 MHz, CDCl3): d 7.92–7.80 (2H,
m, H-20, H-60, AA0BB0), 7.30–7.19 (2H, m, H-30, H-50, AA0BB0),
7.09 (1H, d, J ¼ 8.2 Hz, H-6), 6.46 (1H, d, J ¼ 2.5 Hz, H-3), 6.42
(1H, dd, J ¼ 8.2, 2.5 Hz, H-5), 3.80 (3H, s, C-2-OCH3), 3.79
(3H, s, C-4-OCH3), 3.23–3.09 (2H, m, H-a), 3.02–2.87 (2H, m, H-
b), 2.40 (3H, s, C-40-CH3); 13C NMR (150 MHz, CDCl3): d 199.97
(C]O), 159.57 (C-4), 158.49 (C-2), 143.69 (C-40), 134.67 (C-10),
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130.43 (C-6), 129.31 (C-30, C-50), 128.37 (C-20, C-60), 122.12 (C-1),
3-(4-Carboxyphenyl)-1-(4-methylphenyl)propan-1-ol
(8b)
103,95 (C-5), 98.67 (C-3), 55.50 (C-2-OCH3), 55.35 (C-4-OCH3), white solid, mp 134–137 ꢀC; [a]D20¼ ꢂ2.733 (c ¼ 0.9; CHCl3);
39.26 (C-a), 25.34 (C-b), 21.76 (C-40-CH3); HR ESI-MS m/z FTIR-ATR (cmꢂ1): 3541.71, 2928.77, 2852.96, 1681.48, 1610.44,
calculated for C18H21O3 [M + H]+ 285.1485, found [M + H]+ 1575.23, 1512.98, 1423.63, 1314.93, 1289.47, 1174.00, 1063.42,
285.1486.
925.05, 814.95, 765.85; 1H NMR (600 MHz, CDCl3): d 8.01 (2H, d,
4-Ethyl-40-methyl-a,b-dihydrochalcone (5a), white solid, mp J ¼ 8.2 Hz, H-3, H-5), 7.28 (2H, d, J ¼ 7.9 Hz, H-2, H-6), 7.24 (2H,
36–38 ꢀC; FTIR-ATR (cmꢂ1): 2971.60, 2921.82, 1679.29, 1604.56, d, J ¼ 7.9 Hz, H-20, H-60), 7.17 (2H, d, J ¼ 7.8 Hz, H-30, H-50), 4.65
1574.48, 1514.73, 1441.94, 1406.62, 1360.86, 1289.24, 1201.94, (1H, dd, J ¼ 7.8, 5.4 Hz, –CH–OH), 2.84–2.77 (1H, m, H-b), 2.77–
1
1184.31, 1059.99, 990.29, 971.67, 819.19, 767.02; H NMR (600 2.69 (1H, m, H-b), 2.35 (3H, s, C-40-CH3), 2.19–2.11 (1H, m, H-a),
MHz, CDCl3): d 7.88 (2H, d, J ¼ 8.2 Hz, H-20, H-60), 7.26 (2H, d, J 2.07–1.98 (1H, m, H-a); 13C NMR (150 MHz, CDCl3): d 171.83
¼ 8.2 Hz, H-30, H-50), 7.19 (2H, d, J ¼ 8.1 Hz, H-2, H-6), 7.15 (2H, (COOH), 148.54 (C-10), 141.39 (C-1), 137.67 (C-40), 130.50 (C-3, C-
d, J ¼ 8.1 Hz, H-3, H-5), 3.30–3.24 (2H, m, H-a), 3.08–3.01 (2H, 5), 129.41 (C-30, C-50), 128.74 (C-2, C-6), 127.24 (C-4), 126.00 (C-
m, H-b), 2.64 (2H, q, J ¼ 7.6 Hz, –CH2CH3), 2.42 (3H, s, C-40- 20, C-60), 73.75 (–CH–OH), 40.04 (C-a), 32.34 (C-b), 21.27 (C-40-
CH3), 1.24 (3H, t, J ¼ 7.6 Hz, –CH2CH3); 13C NMR (150 MHz, CH3); HR ESI-MS m/z calculated for C17H17O3 [M ꢂ H]ꢂ
CDCl3): d 199.12 (C]O), 143.88 (C-40), 142.12 (C-4), 138.67 (C-1), 269.1172, found [M ꢂ H]ꢂ 269.1178.
134.54 (C-10), 129.37 (C-30, C-50), 128.46 (C-2, C-6), 128.28 (C-20,
C-60), 128.10 (C-3, C-5), 40.60 (C-a), 29.94 (C-b), 28.57
Antimicrobial activity
(–CH2CH3), 21.74 (C-40-CH3), 15.78 (–CH2CH3); HR ESI-MS m/z
calculated for C18H21O [M + H]+ 253.1587, found [M + H]+ Broad spectrum of biological properties of chalcones and their
253.1588.
derivatives is associated with the diversity of these polyphenolic
4-Ethoxy-40-methyl-a,b-dihydrochalcone (6a), white solid, compounds. Chalcones and dihydrochalcones belong to
mp 68–70 ꢀC; 1H NMR (600 MHz, CDCl3): d 7.89–7.83 (2H, m, H- secondary plant metabolites and constitute a natural protective
20, H-60, AA0BB0), 7.26–7.23 (2H, m, H-30, H-50, AA0BB0), 7.18–7.13 barrier against microbial infections. Phloretin is one of the
(2H, m, H-2, H-6, AA0BB0), 6.86–6.80 (2H, m, H-3, H-5, AA0BB0), most popular dihydrochalcones exhibiting strong antimicrobial
4.01 (2H, q, J ¼ 7.0 Hz, –OCH2CH3), 3.27–3.21 (2H, m, H-a), activity against Erwinia amylovora and Venturia inaequalis –
3.03–2.97 (2H, m, H-b), 2.41 (3H, s, C-40-CH3), 1.40 (3H, t, J ¼ Gram-negative bacteria and fungus, which are a common cause
7.0 Hz, –OCH2CH3); 13C NMR (150 MHz, CDCl3): d 199.22 (C] of apple diseases.28
O), 157.44 (C-4), 143.90 (C-40), 134.57 (C-10), 133.41 (C-1), 129.44
As a result of our investigations, antimicrobial activity of
(C-2, C-6), 129.39 (C-20, C-60), 128.30 (C-30, C-50), 114.66 (C-3, C- synthesised chalcones (1–8) and biotransformation products
5), 63.56 (–OCH2CH3), 40.75 (C-a), 29.53 (C-b), 21.76 (C-40-CH3), (1a–8a, 8b) was characterized. Inhibitory effect of all
15.02 (–OCH2CH3); HR ESI-MS m/z calculated for C18H21O2 [M + compounds was evaluated against two strains of bacteria and
H]+ 269.1536, found [M + H]+ 269.1539.
four strains of fungi (Tables 2 and 3).
40-Methyl-4-nitro-a,b-dihydrochalcone (7a), white solid,
Previously, Stompor et al. tested antibacterial activity of 40-
mp97–100 ꢀC (lit.27 104–105 ꢀC); 1H NMR (600 MHz, CDCl3): methoxychalcone derivatives against E. coli PCM2560, S. aureus
d 8.17–8.12 (2H, m, H-3, H-5, AA0BB0), 7.87–7.82 (2H, m, H-20, H- PCM2054 and C. albicans KL-1 using agar diffusion method
60, AA0BB0), 7.44–7.39 (2H, m, H-2, H-6, AA0BB0), 7.26 (2H, d, J ¼ with 10 mL of 20% (m/v) compounds applied onto each test disc.
8.5, H-30, H-50), 3.32 (2H, t, J ¼ 7.4 Hz, H-a), 3.18 (2H, t, J ¼ They reported, that chalcones with methoxy group attached to
7.4 Hz, H-b), 2.41 (3H, s, C-40-CH3); 13C NMR (150 MHz, CDCl3): the C-40 position had no bacteriostatic effect on E. coli
d 197.91 (C]O), 149.45 (C-4), 146.63 (C-40), 144.39 (C-1), 134.18 PCM2560.29 As a result of our study, complete inhibition of
(C-10), 129.52 (C-2, C-6), 129.49 (C-30, C-50), 128.24 (C-20, C-60), growth of E. coli ATCC10536 in presence of compounds 1–6 and
123.88 (C-3, C-5), 39.41 (C-a), 29.96 (C-b), 21.80 (C-40-CH3); HR
8
was observed. Contrarily, 40-methyl-4-nitrochalcone (7)
ESI-MS m/z calculated for C16H16NO3 [M + H]+ 270.1125, found enabled the limited growth of this strain of bacteria (DOD ¼
[M + H]+ 270.1128.
0.31) and additionally caused prolongation of the lag phase
4-Carboxy-40-methyl-a,b-dihydrochalcone (8a), white solid, from 4 to 22 hours. Sivakumar et al. calculated the Minimal
mp 142–145 C; FTIR-ATR (cmꢂ1): 2920.80, 2850.12, 1676.92, Inhibitory Concentration (MIC) of 4-methoxy-40-methylchalcone
ꢀ
1608.75, 1574.89, 1426.10, 1403.52, 1315.66, 1291.75, 1236.27, against S. aureus NCIM5021 at the level of 0.248 mM. Unfortu-
1
1173.49, 1101.72, 1043.14, 972.35, 931.83, 811.41, 769.06; H nately, this activity is three times weaker than the popular
NMR (600 MHz, CDCl3): d 8.04 (2H, d, J ¼ 8.2 Hz, H-3, H-5), antibiotic ampicillin.30 In our research, 40-methylchalcone
7.86 (2H, d, J ¼ 8.2 Hz, H-20, H-60), 7.35 (2H, d, J ¼ 8.2 Hz, H- derivatives with alkyl, O-alkyl or nitro group attached to the C-4
2, H-6), 7.25 (2H, d, J ¼ 8.2 Hz, H-30, H-50), 3.31 (2H, t, J ¼ position, also exhibited a very weak effect considering limita-
7.6 Hz, H-a), 3.14 (2H, t, J ¼ 7.6 Hz, H-b), 2.41 (3H, s, C-40-CH3); tion of S. aureus DSM799 growth, but they prolonged the
13C NMR (150 MHz, CDCl3): d 198.48 (C]O), 172.01 (COOH), adaptive phase from 3 to 23.5–26.0 hours. However, 2,4-dime-
148.03 (C-40), 144.20 (C-1), 134.33 (C-10), 130.63 (C-3, C-5), thoxy-40-methylchalcone (4) exhibited activity that was twice as
129.47 (C-30, C-50), 128.75 (C-2, C-6), 128.29 (C-20, C-60), strong as the chalcone without methoxy group attached to the
127.47 (C-4), 39.78 (C-a), 30.31 (C-b), 21.79 (C-40-CH3); HR ESI- C-2 position (3). A similar effect was observed for 4-carboxy-40-
MS m/z calculated for C17H17O3 [M + H]+ 269.1172, found [M + methylchalcone, which also extended the lag phase of S. aureus
´
H]+ 269.1179.
DSM799. Lopez et al. reported that 4-methoxy-40-
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Table 2 Antimicrobial activity of 40-methylchalcones (1–8)a
E. coli
S. aureus
C. albicans
A. alternata
F.linii
A. niger
Strain
ATCC10536
DSM799
DSM1386
CBS1526
KB-F1
DSM1957
Control
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
4.0
1.62
—
0.0
—
0.0
—
0.0
—
0.0
—
0.0
—
0.0
22.0
0.31
—
3.0
1.66
23.5
1.42
26.0
1.22
24.0
1.08
5.0
0.46
24.5
1.30
24.0
1.04
26.0
1.02
28.5
0.45
3.0
1.56
5.0
1.54
6.0
1.53
4.5
1.32
7.5
0.63
5.0
1.63
5.0
1.19
6.5
1.02
16.5
0.29
16.5
1.85
26.0
1.03
19.0
0.83
18.0
0.82
36.5
0.23
20.0
1.06
19.5
0.68
34.0
0.93
—
13.5
1.93
3.5
1.34
2.5
0.96
2.5
1.14
9.0
0.75
3.5
1.33
3.0
1.03
6.0
0.93
3.5
10.5
2.13
27.0
0.91
33.5
0.70
20.5
0.69
35.5
0.28
20.5
1.12
20.5
0.74
34.0
1.08
—
1
2
3
4
5
6
7
8
Lag-phase [h]
DOD
0.0
0.0
0.32
0.0
a
OD – optical density (l ¼ 560 nm).
methylchalcone (3) exhibited strong inhibitory activity against identied. Furthermore, 2,4-dimethoxy-40-methylchalcone
two dermatophytes fungi Microsporum canis C112 and Epi- limited the proliferation of C. albicans DSM1386 (DOD ¼ 0.63).
dermophyton occosum C114 with the MIC value of 3 mg mLꢂ1 As a result of 4-carboxy-40-methylchalcone action, high inhibi-
and 0.5 mg mLꢂ1, respectively. In contrast, antifungal activity tion of C. albicans DSM1386 (DOD ¼ 0.29) and F. linii KB-F1
against C. albicans, Saccharomyces cerevisiae, Cryptococcus neo- (DOD ¼ 0.32) growth was observed. Moreover, this derivative
formans, Aspergillus niger, A. fumigatus and A. avus was not provided total inhibition of growth of A. alternata CBS1526 and
observed.31 In the case of C. albicans DSM1386 we noted the A. niger DSM1957.
same lack of response to the chalcone 3 and also to the other
In the literature, there is no information about biological
alkyl (methyl and ethyl) derivatives (1, 2, 5). Partial growth activity of compounds 1a–8a and 8b. As a result of our investi-
inhibition in the presence of 4-ethoxy-40-methylchalcone was gation, all dihydrochalcones (1a–8a) hindered growth of E. coli
Table 3 Antimicrobial activity of products of biotransformations (1a–8a, 8b)a
E. coli
S. aureus
C. albicans
A. alternata
F.linii
A. niger
Strain
Control
1a
ATCC10536
DSM799
DSM1386
CBS1526
KB-F1
DSM1957
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
Lag-phase [h]
DOD
4.0
1.61
16.5
0.17
15.5
0.15
15.5
0.24
15.0
0.31
15.5
0.30
4.5
0.44
14.5
0.10
14.0
0.10
—
2.5
1.67
4.0
0.54
7.5
0.56
6.0
0.51
7.5
0.48
8.0
0.43
2.5
0.63
6.0
3.0
1.58
8.0
0.61
5.0
0.78
8.0
0.69
8.5
0.61
12.0
0.74
7.0
0.82
8.0
0.53
14.0
0.30
21.0
0.64
16.5
1.86
18.0
0.74
16.5
1.20
18.5
1.07
17.0
0.96
25.0
1.49
18.0
1.28
17.5
0.77
24.0
0.34
18.5
0.51
13.5
1.95
14.5
1.21
9.5
11.0
2.14
29.0
0.77
30.0
0.92
29.0
0.63
31.5
0.99
31.5
0.97
28.0
0.89
35.5
0.94
—
2a
1.08
14.5
1.19
13.0
1.21
13.0
1.27
12.0
1.17
10.0
0.94
11.0
0.24
21.0
0.67
3a
4a
5a
6a
7a
0.48
18.0
0.19
10.5
0.45
8a
Lag-phase [h]
DOD
Lag-phase [h]
DOD
0.0
35.0
0.91
8b
0.0
a
OD – optical density (l ¼ 560 nm).
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ATCC10536 and prolonged its lag phase. Furthermore, 3-(4- and A. niger DSM1957 chalcones and dihydrochalcones
carboxyphenyl)-1-(4-methylphenyl)propan-1-ol (8b) prevented demonstrated similar activity, excluding 4-carboxy-40-methyl-
growth of this bacterial strain. In the case of S. aureus DSM799 a,b-dihydrochalcone (8a), which caused complete inhibition
growth, dihydrochalcones with alkyl group attached to the C-4 of growth A. niger DSM1957 (DOD ¼ 0).
(1a, 2a, 5a) had inhibitory effect 3 times stronger than corre-
sponding chalcones (1, 2, 5). Furthermore, biotransformation
products with O-alkyl chains at the C-4 position (3a, 6a)
Conflicts of interest
exhibited 2 times stronger activity in comparison to their chal-
cones (3, 6). Only, 2,4-dimethoxy-40-methyl-a,b-dihydrochalcone
The authors declare no conict of interest.
(4a) showed inhibitory effect similar to its chalcone (4). More-
over, 3-(4-carboxyphenyl)-1-(4-methylphenyl)propan-1-ol (8b) Acknowledgements
exhibited the same strength of action but shortened the lag
Publication supported by the National Science Centre, Grant
phase from 28.5 to 10.5 hours in comparison to its chalcone (8).
No. 2016/21/B/NZ9/01904 and the Wroclaw Centre of Biotech-
In addition, dihydrochalcone with carboxyl group attached to
nology, programme the Leading National Research Centre
the C-4 position showed 2 times stronger inhibitory activity
(KNOW) for years 2014–2018.
than the corresponding chalcone (8) and alcohol (8b). In the
case of C. albicans DSM1386, alkyl, O-alkyl and nitro dihy-
drochalcone derivatives (1a–3a, 5a–7a) showed antifungal
activity about 3 times stronger than the chalcones (1–3, 5–7).
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