Antimicrobial activity of xanthohumol and its selected structural analogues

Article abstract of DOI:10.3390/molecules21050608

The objective of this study was to evaluate the antimicrobial activity of structural analogues of xanthohumol 1, a flavonoid compound found in hops (Humulus lupulus). The agar-diffusion method using filter paper disks was applied. Biological tests performed for selected strains of Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, fungi (Alternaria sp.), and yeasts (Rhodotorula rubra, Candida albicans) revealed that compounds with at least one hydroxyl group - all of them have it at the C-4 position - demonstrated good activity. Our research showed that the strain S. aureus was more sensitive to chalcones than to the isomers in which the heterocyclic ring C is closed (flavanones). The strain R. rubra was moderately sensitive to only one compound: 4-hydroxy-4′-methoxychalcone 8. Loss of the hydroxyl group in the B-ring of 4′-methoxychalcones or its replacement by a halogen atom (-Cl, -Br), nitro group (-NO₂), ethoxy group (-OCH₂CH₃), or aliphatic substituent (-CH₃, -CH₂CH₃) resulted in the loss of antimicrobial activity towards both R. rubra yeast and S. aureus bacteria. Xanthohumol 1, naringenin 5, and chalconaringenin 7 inhibited growth of S. aureus, whereas 4-hydroxy-4′-methoxychalcone 8 was active towards two strains: S. aureus and R. rubra.

Full text of DOI:10.3390/molecules21050608

molecules
Article
Antimicrobial Activity of Xanthohumol and
Its Selected Structural Analogues
3
Monika Stompor 1,2,* and Barbara Zarowska
˙
1
Department of Chemistry, University of Environmental and Life Sciences, Norwida 25,
50-375 Wrocław, Poland
2
Centre for Innovative Research in Medical and Natural Sciences, Faculty of Medicine, University of Rzeszów,
Warzywna 1a, 35-310 Rzeszów, Poland
Department of Biotechnology and Food Microbiology, University of Environmental and Life Sciences,
3
Chełmon´skiego 37/41, 51-630 Wrocław, Poland; barbara.zarowska@up.wroc.pl
*
Correspondence: monika.stompor@gmail.com; Tel.: +48-17-851-6855
Academic Editors: Celestino Santos-Buelga and Arturo San Feliciano
Received: 30 March 2016; Accepted: 5 May 2016; Published: 11 May 2016
Abstract: The objective of this study was to evaluate the antimicrobial activity of structural analogues
of xanthohumol
1, a flavonoid compound found in hops (Humulus lupulus). The agar-diffusion
method using filter paper disks was applied. Biological tests performed for selected strains of
Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, fungi (Alternaria sp.)
and yeasts (Rhodotorula rubra, Candida albicans) revealed that compounds with at least one hydroxyl
group—all of them have it at the C-4 position—demonstrated good activity. Our research showed
that the strain S. aureus was more sensitive to chalcones than to the isomers in which the heterocyclic
ring C is closed (flavanones). The strain R. rubra was moderately sensitive to only one compound:
,
4-hydroxy-4’-methoxychalcone
its replacement by a halogen atom (
or aliphatic substituent ( CH3, CH2CH3) resulted in the loss of antimicrobial activity towards
8. Loss of the hydroxyl group in the B-ring of 4’-methoxychalcones or
´
Cl, Br), nitro group ( NO2), ethoxy group ( OCH2CH3),
´
´
´
´
´
both R. rubra yeast and S. aureus bacteria. Xanthohumol
inhibited growth of S. aureus, whereas 4-hydroxy-41-methoxychalcone
strains: S. aureus and R. rubra.
1
, naringenin
5, and chalconaringenin
7
8
was active towards two
1
Keywords: antimicrobial activity; xanthohumol; chalconaringenin; naringenin; 4 -methoxychalcones;
S. aureus; NMR-IR-UV spectra
1. Introduction
There is a growing interest in chemical compounds of natural origin having antimicrobial
properties, which gives rise to a number of studies in this field. Current investigations into the
antimicrobial activity of flavonoid compounds have targeted plant extracts, pure compounds, products
of their metabolism, and also their synthetic analogues [
of all groups of flavonoid compounds, are more often recommended to combat different kinds of
intestinal parasites that cause food poisoning in humans, for example Giardia lamblia [ ]. They are
also used as acetylcholinesterase inhibitors in the treatment of neurodegenerative diseases such as
Alzheimer’s [ ]. Additionally, chalcones demonstrate cytotoxic activity towards human cancer cells [ ].
A valuable source of flavonoids and phenolic acids is beer, which is commonly consumed in the
majority of countries all over the world [ ]. Analysis of different brands of beer of Czech, Slovakian,
Romanian, and Serbian origin revealed that it contains genistein, biochanin, daidzein, formononetin,
quercetin, and apigenin [ ]. Moreover, due to the addition of hops, beer contains such flavonoids as
1]. Nowadays chalcones which are precursors
2
3
4
5
6,7
Molecules 2016, 21, 608; doi:10.3390/molecules21050608
www.mdpi.com/journal/molecules

Molecules 2016, 21, 608
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xanthohumol
1, isoxanthohumol
2, 8-prenylnaringenin
3, trace amounts of
α
,β-dihydroxanthohumol
4 [8], naringenin 5, and their derivatives (Figure 1).
CH3
H3C
HO
CH3
H3C
HO
CH3
H3C
OH
OH
OH
OH
O
O
HO
O
O
O
O
O
OH
H3C
H3C
(1)
(2)
(3)
CH3
H3C
HO
OH
HO
OH
OH
HO
O
OH
OH
OH
O
OH
O
O
O
H3C
(4)
(5)
(6)
OH
HO
OH
OH
O
(7)
Figure 1.
Chemical structures of xanthohumol
1
and its derivatives: isoxanthohumol
2,
8-prenylnaringenin
3,
α
,
β-dihydroxanthohumol
4
, naringenin -dihydrochalconaringenin , and
5
,
α
,
β
6
chalconaringenin 7.
In Poland, the best source of
content of this prenylated chalcone is estimated at up to 0.22 mg/L and is dependent on the amount of
hop extract [ ]. Germany has developed a technology for production of beer enriched in xanthohumol
(“XAN technology”). It allows xanthohumol concentratiosn of 1–3 mg/L in non-filtered lager-type
beer and over 10 mg/L in filtered dark beer. In the Czech Republic “ZATEC Xantho” beer contains
0.3 mg/L of 10]. Encapsulated forms of hop extracts with estrogenic activity, rich in flavonoids,
are known in Western Europe under the brand names Xantoflav (Germany), Lifenol, Naturex, and
Avignon (France) [11]. Aromatic oils and bitter hop acids give beer its characteristic aroma and bitter
taste and play a protective role against microbial infections [ 12]. The most recent reports suggest
that there is a possibility of application of hop extract, containing bitter hop acids and , in the meat
1 in the diet is dark beer and light non-filtered beer, in which the
9
1
˙
1
[
™
6,7,
1
industry as a natural preservative that inhibits the development of golden staph (Staphylococcus aureus)
and thus increases food safety [13]. In addition, the antifeedant activity of hop compounds, selected
synthetic chalcone derivatives, and chromones against the peach-potato aphid (Myzus persicae) has
been reported recently [14].
Jayasinghe et al. [15] proved that plant geranylchalcones isolated from Artocarpus nobilis (Moraceae)
have antifungal activity towards Cladosporium cladosporioides. López et al. [16] suggested that
electron-donating substituents (e.g.,
towards human dermatophytes, while electron acceptor groups (e.g.,
the opposite effect and augment this activity.
´
OCH3) in ring B of chalcones decrease their antifungal activity
´NO2) at the para position have

Molecules 2016, 21, 608
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Isolated from Dorstenia barteri (Moraceae), isobavachalcone, which has a structure similar to the
prenylated hop chalcone , inhibited the growth of several pathogenic strains, including bacteria
(IC50 = 0.3 M for Enterobacter cloacae, S. aureus, Bacillus stearothermophilus), filamentous fungi, and
yeast (IC50 = 0.3 M for C. albicans, Candida gabrata). Significant results were obtained for two strains,
1
µ
µ
Microsporum audorium and Trichophyton rubrum, where the measured IC50 value was the same as for
nystatin, used as a reference standard [17].
Flavonoids may indirectly chelate transition metal ions, for example copper and iron, which
prevents formation of the highly reactive hydroxyl radical in cells. Kaveri et al. [18] obtained ruthenium
1
complexes with 2 -hydroxychalcone derivatives, substituted in the para position with a methyl group,
chlorine, and naphthalene. The compounds were tested for antimicrobial activity against S. aureus
and Salmonella enterica. The ruthenium compounds were more active than 21-hydroxychalcone itself.
Higher activity of the chelates was rationalized by the loss of the polar nature of the metal ion, caused
by the delocalization of
π electrons in the ring. Additionally, the lipophilic character of the whole
compound increased, which resulted in better permeability of the conjugates through a phospholipid
bilayer membrane of bacterial cells.
Because of the constant risk of pathogenic bacterial infections, to assess the potential utility
of the obtained compounds as antibacterial and antifungal agents we performed biological tests
of xanthohumol
antimicrobial activity against five microbial strains. The considerably low number of strains tested for
1, naringenin 5, chalconaringenin 7 8–15 for their
, and eight 41-methoxychalcones
1
microbicidal sensitivity to 4 -methoxychalcones (Figure 2) and the lack of information about any kind
1
1
of biological activity of 4-bromo-4 -methoxychalcone 10 and 4-ethyl-4 -methoxychalcone 13 prompted
us to undertake research in this field. To the best of our knowledge, there is no information about the
antimicrobial activity of the rest of the compounds against the chosen microorganisms.
Cl
OH
Br
O
O
O
H3C
H3C
H3C
O
O
O
(8)
(9)
(10)
NO2
CH3
O
O
H3C
H3C
O
O
(11)
(12)
O
CH3
O
H3C
O
(13)
CH3
O
O
H3C
H3C
O
O
(14)
(15)
Figure 2. Chemical structure of 41-methoxychalcones 8–15.

Molecules 2016, 21, 608
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2. Results
Antimicrobial Activity
For the biological tests we chose three hop flavonoids: xanthohumol
1
, chalconaringenin
15, differing in the
electronegativity of the substituents at C-4. The minimal concentration that inhibits growth of a
7, and
naringenin 5 8–
. Additionally, we tested a group of eight 41-methoxychalcones
1
microorganism (MIC) for 4 -methoxychalcone 15, known from the literature, determined towards ten
different microorganisms was equal or more than 100 g/mL [19]. For our tests we decided to use
µ
the disc diffusion method and to add to the group of microorganisms described in that work two
strains that were not tested: Alternaria sp. KK-1 and R. rubra C-9. Additionally, we planned to check
the impact of different substituents in the para position of the B aromatic ring of 41-methoxychalcone
15 on the antimicrobial properties.
Initially, for assessment of the antimicrobial activity we planned to use the turbidimetric method
with the help of the Bioscreen C system. Unfortunately, we observed that when a DMSO solution
of a tested compound was added to the cultivation medium, a precipitate appeared. For this reason
we used the agar disc diffusion method. In the case of substances that are poorly water-soluble,
the drawback of this method is weak diffusion of the compounds into the agar medium, which
was prepared in water [20,21]. For this reason we decided to use high concentrations of the tested
compounds ( 15). However, despite such high doses, we observed that only four of the tested
1, 5, 7, 8–
compounds demonstrated moderate antimicrobial activity, toward only one out of five tested strains.
Some of the strains were totally resistant to the tested agents. Among the tested microorganisms only
S. aureus PCM 2054 bacterium, and R. rubra C-9, yeast, were sensitive to the tested chalcones (Table 1).
Table 1. Antimicrobial activity of tested compounds.
Microorganism
Compound
Number
E. coli
S. aureus
C. albicans
R. rubra
Alternaria sp.
dinh (mm)
(1)
(5)
-
-
3.57 *
3.77 **
1.92 **
6.84 **
-
-
-
-
-
-
(7)
-
-
-
-
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
-
-
-
-
-
-
-
-
2.86 *
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2.46 **
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
oxytetracycline
cyklohexymide
4.17 *
9.18 *
13.21 *
11.32 *
3.91 *
* Zone of inhibition in mm; ** lighter zone of inhibition in mm; “-“no activity.
Among eight 41-methoxychalcones
4-hydroxy-41-methoxychalcone
inhibited the growth of the golden staph S. aureus and yeast
R. rubra (Figure 3). The lack of hydroxyl group or its replacement by a halogen atom ( Cl Br 10),
8–15 tested for antimicrobial activity, only
8
´
9,
´
nitro group (´NO2 11), ethoxy group (´O´CH2CH3 13), or aliphatic groups (´CH2CH3 12), (´CH3 14)
led to inactivation of the compounds with respect to both R. rubra yeast and S. aureus bacterium.

Molecules 2016, 21, 608
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Staphylococcus aureus
Rhodotorula rubra
Figure 3. Influence of selected hydroxychalcones on the growth of the strains: (
) R. rubra: (I) chalconaringenin ( ); (II) xanthohumol (
); (III) 4-hydroxy-41-methoxychalcone (
(K) control (DMSO).
A
) S. aureus,
(
B
7
1
8);
Studies on the relationship between compound structure and its activity towards bacteria revealed
that the strain S. aureus is more sensitive to chalcones, compared to the isomers with a closed
heterocyclic ring C. Chalconaringenin
of S. aureus, which resulted in the appearance of lighter zones around the discs with tested compounds
(with much smaller number of microcolonies) of diameters dinh = 6.84 mm and 3.77 mm, respectively.
7
and ( )-naringenin 5 considerably inhibited the growth
˘
The obtained prenylated hop chalcone-xanthohumol
1 generated the largest inhibition zone of all
tested chalcones in the case of the strain S. aureus (dinh = 3.57 mm).
3. Discussion
The dilution method used by Rozalski et al. [22] in research on
1 with respect to three strains
of S. aureus of different origin led to determination of the range of minimal inhibition concentration
(MIC) as 15.6 to 62.5 g/mL. Our results are consistent with the results of Ávila et al. [23], concerning
evaluation of antimicrobial activity of xanthohumol derivatives: isocordoin (which lacks 4-OH
and C-6’-OCH3 substituents, compared to
) and 4-hydroxyisocordoin (lacking C-61-OCH3 group).
µ
1
1
They proved that the presence of a hydroxyl group at C-4 is crucial for the antimicrobial activity towards
S. aureus. Moreover, replacement of the substituent at C-8 in 4-hydroxyisocordoin with a geranyl one
did not affect the activity towards S. aureus, whereas growth of Bacillus cereus was more effectively
inhibited by the derivative with the geranyl group. On the other hand, cyclization of the isoprenyl
group resulted in a loss of activity. A few years later the observation that the electron-donating group
in the B ring of chalcones has a positive effect on their antimicrobial activity was also confirmed by
Wu et al. [24]. Similarly to our results, Gram-positive bacteria (S. aureus, Staphylococcus epidermidis,
Enterococcus faecalis, Enetrococcus faecium) were more resistant to 4-hydroxy-41-methoxychalcone
8
than Gram-negative ones (E. coli, Klebsiella pneumonia, Pseudomonas aeruginosa). Probably these
differences are related to differences in composition of the cytoplasmic membrane in Gram-positive
and Gram-negative bacteria. Similar assumptions were made in the research on the influence of hop
extract on biochemical processes in Gram-positive and Gram-negative bacteria [25]. Karaman et al. [26
]
proved that substitution of 41-methoxychalcone 15 at the para position in ring B with a chlorine atom
results in deactivation of this compound towards the following pathogens: Proteus vulgaris KUEN
1329, Candida utilis KUEN 1031, and P. aeruginosa ATCC 9027. The activity of 4’-methoxychalcone 15
and 4-chloro-41-methoxychalcone
9
towards Streptomyces pyogenes ATCC 176 was comparable—the
diameters of inhibition zones were 9 and 10 mm, respectively. Conversely, a positive influence of the

Molecules 2016, 21, 608
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chlorine atom in
9
on the antimicrobial activity was observed for the anaerobic streptococcus E. faecalis
1
ATCC 29122 (dinh = 10 mm), which was resistant to non-substituted 4 -methoxychalcone 15.
Our study showed that in the case of the second hop flavonoid, chalconaringenin
7 and
1
4-hydroxy-4 -methoxychalcone
8, the zones of inhibition for S. aureus bacteria were smaller compared to
xanthohumol : 1.92 mm and 2.86 mm, respectively. The research on the structure–activity relationship
1
in chalcone-flavone compounds with respect to bacterial sensitivity to antimicrobials showed that the
strain S. aureus is more sensitive to chalcones than to the isomers in which the heterocyclic ring C is
closed. Our research showed that this statement is also valid in the case of naringenin
is commonly found in citrus fruits, and its isomer, chalconaringenin . Not only did these compounds
considerably inhibit the growth of golden staph, which resulted in a lighter zone with a much lower
5, a flavonoid that
7
number of visible microcolonies, but also chalcone form
7 was more active than the cyclic one,
5 (dinh = 6.84 mm and 3.77 mm, respectively).
As the results show, the concentration of tested substances with respect to their growth inhibition
activity is an important and so far not fully resolved issue. Our results add to the earlier study on
antibiotic activity of naringenin
that application of this flavonoid at a concentration of 250
the growth of the golden staph bacterium. In the case of such strains as Salmonella enteritidis and
B. cereus, the higher concentration of 400 g/mL was also insufficient [28]. By contrast, the microbicidal
activity of towards Gram-negative Acinetocabter haemolyticus bacteria (MIC = 0.183 mmol/mL) was
5
towards S. aureus, reported by Sasaki et al. [27], which showed
µg/mL was not sufficient for inhibiting
µ
5
described by Chatzopoulou et al. [29]. Other strains tested by this group, including Empedobacter brevis,
P. aeruginosa, and K. pneumonia, were resistant to 5.
On the other hand, Shakil et al. [30], when comparing the microbial activity of chalcones and their
isomers (flavanones) came to the conclusion that in the case of microorganisms other than S. aureus
more active were compounds with the closed heterocyclic ring C. Only in the case of the non-substituted
1
flavanone and 2’-hydroxychalcone, and also for 4-bromoflavanone and 4-bromo-2 -hydroxychalcone,
was there no difference in activity toward one of the two tested phytopathogens (Rhizoctonia solani ITCC
5563 and Sclerotium rolfsii ITCC 6181). The results for R. solani were similar for the two isomeric forms.
This indicates that generalizations concerning the structure-activity relationship of the mentioned
compounds cannot be applied to a wide range of microorganisms. The antibacterial activity of
hydroxylated flavanone derivatives, including naringenin
5 towards streptococci E. faecalis, which
is the bacterium most often found in hospitals (where there is usually a high level of resistance to
antibiotics), was also confirmed by the other research group [31].
Moreover, the antimicrobial activity of the tested compounds may be dependent on the
enzymatic system of the microorganisms and possible processes of detoxification. Degradation
of
Herles et al. [32]. This enzyme was isolated from the cells of anaerobic bacteria Eubacterium ramulus
DSM 16296. In the first stage of conversion naringenin underwent isomerization to chalconaringenin
and then, due to hydrogenation of the olefinic bond in the presence of catalytic amounts
of NADH– -dihydrochalconaringenin, was formed. Chalconaringenin also undergoes
biotransformation to phloretin in the cells of aerobic bacteria, such as Rhodococcus and Gordonia,
as we described earlier [33]. The newest study proved the strong antibacterial properties of phloretin
and its glycoside derivatives towards Gram-positive (S. aureus ATCC 6538, Listeria monocytogenes
ATCC 13932) and Gram-negative (Salmonella typhimurium ATCC 133111) bacteria [34]. As we reported
earlier [35], when was subjected to transformation by Rhodotorula marina AM 77, we obtained
-dihydroxanthohumol . Most of the metabolites obtained in the biotransformations of , including
4, have not been tested for antimicrobial activity so far.
Our results showed that the strain R. rubra was moderately sensitive to only one compound:
4-hydroxy-4’-methoxychalcone , which generated a lighter zone of inhibition (with a smaller number
of microcolonies) of 2.46 mm diameter. Loss of the hydroxyl group or its replacement by a halogen
atom ( Cl Br 10), nitro group (–NO2 11), ethoxy group ( OCH2CH3 12), or aliphatic substituent
5 with the help of a chalcone isomerase of bacterial origin had been earlier described by
5
7
α
,
β
6
7
6
6
1
α
,
β
4
1
8
´
9,
´
´

Molecules 2016, 21, 608
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(
´
CH3 14),
´
CH2CH3 13) resulted in the loss of antimicrobial activity towards both R. rubra yeast and
S. aureus bacterium. This is in agreement with the theory put forth by Batovska et al. [1], which states
that the antifungal activity of chalcones depends on the presence of substituents that can easily enter
into a Michael reaction with intracellular thiol groups. In this reaction the addition of thiols to the
electrophilic center (C-β) of unsaturated compounds takes place.
4. Materials and Methods
Reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Merck (Darmstadt,
Germany). The commercially available ( )-naringenin 5 (Sigma-Aldrich) was used. The purity of the
˘
synthesized products (>95%) was established by HPLC chromatography. Analysis were performed
according to the procedure described earlier [33]. The NMR spectra (1H-, 13C-NMR, 13C-DEPT, and
1
correlation spectra: H-1H-COSY, 1H-13C-HMQC, 1H-13C-HMBC) were recorded in a CDCl3 solution
on a Brüker Avance DRX 300 and Brüker Avance II 600 MHz spectrometers (Brüker, Karlsruhe,
Germany). IR spectra (KBr discs) were determined on a Thermo-Nicolet IR300 FT-IR-spectrometer
(Madison, WI, USA). UV spectra were run on a Visible Spectrofotometer Cintra 303, GBC, in methanol.
Melting points (uncorrected) were determined on a Boetius apparatus. Bioscreen C (Lab system Oy,
Helsinki, Finland) has been used in the initial part of the investigations of antimicrobial activity.
4.1. Antimicrobial Agents
4.1.1. Xanthohumol and Chalconaringenin
Xanthohumol
1 was isolated from supercritical carbon-dioxide-extracted hops—a waste residue
remaining after extraction of hops (H. lupulus) with supercritical CO2—received from the Supercritical
Extraction Department of New Chemical Syntheses Institute in Puławy. Xanthohumol was obtained
according to the method described earlier [36]. Spectral data of
The next hop chalcone and a precursor of the prenyl derivatives–chalconaringenin
by alkaline isomerization of racemic ( )-naringenin , with the help of a methanolic KOH solution,
1
were consistent with the literature [35].
7
was obtained
˘
5
according to the modified method of Le Bail et al. [37]. Spectral data of
7 were identical with literature
values [33].
1
4.1.2. (E)-4 -Methoxy-4-Substituted Chalcones
To 41-methoxyacetophenone (12 mmol) dissolved in MeOH (10 mL) a benzaldehyde derivative
(12 mmol) dissolved in MeOH (10 mL) was added portionwise. The reaction mixture was stirred
for 10 min at room temperature and then KOH (1.44 g, 36 mmol) was added. The resulting mixture
was stirred for 14–18 h in the dark. Next the mixture was diluted with three volumes of distilled
water and acidified to pH 4–6 with concentrated HCl. The precipitate was filtered on a Schott
filter, washed with large amounts of distilled water, and dried under a vacuum for 5 h. The crude
product was purified by column chromatography on silica gel (eluent: 10%–20% v/v of acetone in
hexane). A series of respective (E)-1,3-diphenylpropen-1-one derivatives was obtained in 60%–85%
yield (see Supplementary Materials).
By this method we obtained 41-methoxychalcone 15 and its seven 41-methoxy para-substituted
derivatives 8–14. In order to introduce substituents of a different chemical nature at the C-4 position, as
substrates we used 4-methoxyacetophenone and benzaldehyde or its derivatives, as follows:
4-chlorobenzaldehyde, 4-bromobenzaldehyde, 4-hydroxybenzaldehyde, 4-nitrobenzaldehyde,
4-methylbenzaldehyde 4-ethylbenzaldehyde, and 4-ethoxybenzaldehyde. The products obtained
were purified and characterized by means of spectral methods (NMR, IR) and spectrophotometric
analysis (UV).

Molecules 2016, 21, 608
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4.2. Evaluation of Antimicrobial Activity
Evaluation of antimicrobial activity of selected compounds
1,
5
,
7
, and
8–
15 were performed
against the strains of bacteria (Escherichia coli PCM 2560 and S. aureus PCM 2054), yeasts (C. albicans KL-1
and Rhodotorula rubra C-9), and filamentous fungi (Alternaria sp. KK-1). The analyses were carried out
by the agar diffusion method using filter paper disks. For the study we used the microorganisms from
the collection of pure cultures of the Department of Biotechnology and Food Microbiology at Wrocław
University of Environmental and Life Sciences and from the Polish Collection of Microorganisms
(PCM), Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy
of Sciences, Wrocław, Poland.
YM agar (for yeasts and fungi) and nutrient broth (for bacteria) were inoculated with a cell
suspension of the selected microorganisms (standardized with a Thoma chamber for yeasts or with
McFarland standards for bacteria) to give a final concentration of 5
of the plates prepared in this way sterile filter-paper discs of 0.5 cm diameter were placed and then
portions of 10 L of 20% (m/v) solution of a tested compound in DMSO were transferred onto the
ˆ
105 CFU/mL. On the surface
µ
discs. At the same time control samples were prepared in an analogous manner, but in this case only
DMSO was applied on the discs. As reference standards we used oxytetracycline (for bacteria) and
cycloheximide (for yeasts and filamentous fungi), which were added at the same concentration as the
tested compounds. The plates were left for 8 h at 4 ˝C to enable diffusion of the tested compounds
to the cultivation medium, and then incubated at 25 ˝C (filamentous fungi) or at 30 ˝C (yeasts and
bacteria) for 48–96 h. A clear zone of inhibition with no microcolonies observed around the disc of
a tested compound was interpreted as its ability to inhibit growth of the microorganism inoculated
in the medium. A visibly lighter zone of inhibition, with a smaller number of microcolonies, was
interpreted as the capability to partially inhibit the growth of a tested strain.
5. Conclusions
The aim of the study was to search for antimicrobial compounds in the group of natural flavonoids
contained in hops (Humulus lupulus) and their synthetic derivatives. The 4’-methoxychalcones (8–15)
were obtained in order to evaluate the impact of compound structures, including electron donating and
electron acceptor substituents at para position in ring B on antimicrobial activity of these compounds.
The strain S. aureus was sensitive toward four out of eleven tested compounds. These compounds
contain at least one hydroxyl group (
compound towards tested Gram-positive bacteria was xanthohumol (
1
,
5
,
7
,
8
), and all of them have it at C-4 position. The most active
), for which inhibition zone
1
diameter in the case of S. aureus was dinh = 3.57 mm. The strain R. rubra was sensitive to only one
compound: 4-hydroxy-4’-methoxychalcone (8).
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/
1420-3049/21/5/608/s1.
Acknowledgments: This work was financially supported by the National Science Centre (Grant No.
2011/01/B/NZ9/02890), 2011–2014. Publication was supported by Wroclaw Centre of Biotechnology, programme
The Leading National Research Centre (KNOW) for years 2014–2018. We would like to thank Mirosław Anioł
from Wrocław University of Environmental and Life Sciences for his comments and suggestions, which helped us
to considerably improve the manuscript.
Author Contributions: Monika Stompor suggested the idea of the investigations, obtained, purified, and
characterized all compounds for biological assays, contributed to the discussion of results, and wrote the paper.
˙
Barbara Zarowska measured the antimicrobial activity. All authors read and approved the final manuscript.
Conflicts of Interest: The authorsdeclare no conflict of interest.
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Sample Availability: Samples of the compounds 1, 5, 7, 8–15 are available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).