Fungal metabolites of xanthohumol with potent antiproliferative activity on human cancer cell lines in vitro

Article abstract of DOI:10.1016/j.bmc.2013.01.026

Xanthohumol (1) and xanthohumol D (2) were isolated from spent hops. Isoxanthohumol (3) was obtained from xanthohumol by isomerisation in alkaline solution. Six metabolites were obtained as a result of transformation of xanthohumol (1) by selected fungal cultures. Their structures were established on the basis of their spectral data. One of them: 2″- (2′′′-hydroxyisopropyl)-dihydrofurano-[4″,5″: 3′,4′]-4′,2-dihydroxy-6′-methoxy-α, β-dihydrochalcone (6) has not been previously reported in the literature. The antioxidant properties of hops flavonoids and xanthohumol derivatives were investigated using the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method. The effects of these compounds on proliferation of MCF-7, PC-3 and HT-29 human cancer cell lines were determined by the SRB assay. With the exception of one metabolite, all tested compounds showed antiproliferative activity against the tested human cancer lines. α,β- Dihydroxanthohumol (4), obtained through the biotransformation of xanthohumol, showed higher antiproliferative activity against MCF-7 human breast carcinoma cell line than cisplatin, a widely used anticancer therapeutic agent, and a comparably high activity against PC-3 human prostate cancer cell line.

Full text of DOI:10.1016/j.bmc.2013.01.026

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fungal metabolites of xanthohumol with potent antiproliferative
activity on human cancer cell lines in vitro
a
b
b
a
Tomasz Tronina a, , Agnieszka Bartmanska , Beata Filip-Psurska , Joanna Wietrzyk , Jarosław Popłonski ,
⇑
´
´
Ewa Huszcza a
a Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
b Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Department of Experimental Oncology, Weigla 12, 53-114 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Xanthohumol (1) and xanthohumol D (2) were isolated from spent hops. Isoxanthohumol (3) was
obtained from xanthohumol by isomerisation in alkaline solution. Six metabolites were obtained as a
result of transformation of xanthohumol (1) by selected fungal cultures. Their structures were estab-
lished on the basis of their spectral data. One of them: 200-(2000-hydroxyisopropyl)-dihydrofurano-
Received 3 October 2012
Revised 7 January 2013
Accepted 11 January 2013
Available online xxxx
[400,500:30,40]-40,2-dihydroxy-60-methoxy-
a,b-dihydrochalcone (6) has not been previously reported in
the literature. The antioxidant properties of hops flavonoids and xanthohumol derivatives were investi-
gated using the 2,20-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method. The effects of these
compounds on proliferation of MCF-7, PC-3 and HT-29 human cancer cell lines were determined by
the SRB assay. With the exception of one metabolite, all tested compounds showed antiproliferative
Keywords:
Xanthohumol
Biotransformation
Antiproliferative activity
Human carcinoma
Antioxidant activity
activity against the tested human cancer lines. a,b-Dihydroxanthohumol (4), obtained through the bio-
transformation of xanthohumol, showed higher antiproliferative activity against MCF-7 human breast
carcinoma cell line than cisplatin, a widely used anticancer therapeutic agent, and a comparably high
activity against PC-3 human prostate cancer cell line.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
xanthohumol, even though its biological properties are less
promising.1 Often cited feature is its function as a precursor of
Hop cones, the female inflorescence of the hop plant used as a
flavouring agent providing bitterness in beer, contain several valu-
able polyphenols, for example, prenylflavonoids and hop bitter
acids. Xanthohumol (30-[300,300-dimethyl allyl]-20,4,40-trihydroxy-
60-methoxychalcone) is the principal prenylated flavonoid found
in hop resin. Recent reports have shown the potential health
benefits of this compound. Xanthohumol exhibits antioxidant,1–3
anti-inflammatory,4 antibacterial,5,6 antiviral,6–8 antifungal6,9 and
antiplasmodial10–12 activities. It has also preventive effect against
osteoporosis13 (see Fig. 1).
8-prenylnaringenin, the most potent phytoestrogen known to
date.21
Given the health promoting potential and good availability of
xanthohumol from sources such as CO2-extracted hops, a waste
product of the hop-processing industry, it might serve as an inter-
esting lead compound. In our research, we used xanthohumol as an
ideal starting material for microbial transformations, a standard
technology for production of compounds which are difficult to ob-
tain by chemical methods.
One of the most important findings have been the potential can-
cer chemopreventive properties of xanthohumol, relevant to sup-
pression of cancer development at the initiation, promotion and
progression phases. Xanthohumol has been shown to inhibit the
growth of a wide variety of human cancer cell lines, including
breast, colon, prostate, ovarian cancer and leukemia.14–20
During the brewing process of beer, xanthohumol is converted
into the corresponding isomeric prenylflavanone isoxanthohumol,
which thus becomes the principal dietary prenylated flavonoid.
2. Materials and methods
2.1. Materials
Xanthohumol (1), the substrate used for the biotransformation
experiments, was isolated from supercritical carbon dioxide ex-
tracted hops (‘Marynka’, crop 2011), obtained from Production of
Hop Extracts (Fertilizer Research Institute, Puławy, Poland) accord-
ing to Stevens et al.22 Xanthohumol obtained by us was identical
with the standard (Alexis Biochemicals, Switzerland).
Isoxanthohumol has
a
significantly better solubility than
Xanhohumol D (2) was obtained by the same method as xan-
1
thohumol (1). H NMR (600 MHz, DMSO-d6) d: 1.72 (3H, s, H-500),
⇑
Corresponding author. Tel.: +48 713205197; fax: +48 3207744.
E-mail address: tomasz.tronina@up.wroc.pl (T. Tronina).
2.64 (1H, dd, J = 6.9, 13.3 Hz, H-100a), 2.76 (1H, dd, J = 6.5, 13.3 Hz,
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.026
Please cite this article in press as: Tronina, T.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.01.026

2
T. Tronina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
H C
3
CH
2
H C
CH
H C
CH
3"
3
3
4"
3
3"
3
5"
5"
4"
5'
5
OH
2"
2"
OH
OH
4'
6'
6
1"
1"
8
OH
OH
4
3
3'
HO
HO
OH
O
HO
O
9
4'
3'
1
1'
7
6
2
3
2'
β
2'
2
α
5'
1'
10
4
5
6'
O
O
O
O
O
CH
CH
CH
3
3
3
1
3
2
H C
3
CH
3
HO
HO
H C
CH
3
CH
3
2"'
3"'
5
OH
OH
OH
H C
4
3
3
6
3"
3'
1"'
2"
O
O
HO
OH
O
OH
OH
O
3
1
4'
5'
β
2'
2
α
1'
6'
O
O
O
O
CH
3
CH
CH
3
3
5
6
4
OH
HO
HO
H C
2"'
CH
H C
CH
3
OH
4'
CH
3
3
3
3"'
5'
H C
3
OH
OH
2"
1"'
3
3"
7
6'
OH
3'
O
O
HO
OH
O
8
O
1'
6
2'
2
β
5
9
3
4
O
O
O
O
O
O
CH
CH
CH
3
3
3
7
8
9
Figure 1. Hop flavonoids (compounds 1–3) and products of xanthohumol (1) biotransformation (compounds 4–9).
H-100b), 3.87 (3H, s, C60O–CH3), 4.22 (1H, m, H-200), 4.60 (1H, s,
H-400a), 4.64 (1H, s, H-400b), 6.06 (1H, s, H-50), 6.84 (2H, m, H-3,
H-5), 7.57 (2H, m, H-2, H-6), 7.66 (1H, d, J = 15.5 Hz, H-b), and
133.9 (C-1), 156.5 (C-4), 162.6 (C-60), 163.7 (C-40), 165.7 (C-20),
and 206.1 (C@O); HR ESI-MS m/z: 357.1755 [M+H]+ (calcd for
C
21H24O5 + H, 357.1697); UV (MeOH) kmax: 292.7 nm.
7.76 (1H, d, J = 15.5 Hz, H-
a
); 13C NMR (151 MHz, DMSO-d6) d:
200-(2000-Hydroxyisopropyl)-dihydrofurano-[400,500:30,40]-40,2-
17.4 (C-500), 28.9 (C-100), 73.7 (C-200), 91.1 (C-50), 104.5 (C-10),
dihydroxy-60-methoxy-
a,b-dihydrochalcone (6) was isolated from
105.2 (C-30), 109.9 (C-400), 116.0 (C-3, C-5), 123.8 (C-
a), 126.0 (C-
Fusarium avenaceum AM11 culture after a 7-day transformation
of xanthohumol (1) (32.8 mg, yield 28%) as well as and from Fusar-
ium oxysporum UPF727 culture after a 16-day transformation of
xanthohumol (1) (12.86 mg, yield 10.2%).
1), 130.6 (C-2, C-6), 142.6 (C-b), 148.1 (C-300), 160.0 (C-4), 160.7
(C-60), 163.4 (C-40), 165.2 (C-20), and 191.7 (C@O); HR ESI-MS m/
z: 393.1311 [M+Na]+ (calcd for C21H22O6Na + H+, 393.1309); ½a D20
ꢀ
+0.894 (c 0.165, MeOH); UV (MeOH) kmax: 368.1 nm. The spectral
data of this compound closely correspond to the literature data
for xanthohumol D.23
1H NMR (600 MHz, acetone-d6) d: 1.22 (3H, s, H-1000), 1.25 (3H, s,
H-3000), 2.85 (2H, m, H-b), 3.01(1H, dd, J = 15.1, 9.6 Hz, H-300a), 3.07
(2H, dd, J = 15.1, 7.9 Hz, H-300b), 3.24 (2H, m, H- ), 3.91 (3H, s, C60-
a
Isoxanthohumol (3) was obtained by the chemical conversion of
OCH3), 4.75 (1H, dd, J = 9.6, 7.9 Hz, H-200), 6.06 (1H, s, H-50), 6.76
(2H, m, H-3, H-5), 7.09 (2H, m, H-2, H-6) and 14.04 (C20-OH); 13C
NMR (151 MHz, acetone-d6) d: 25.4 (C-3000), 26.0 (C- 1000), 27.3
24
´
xanthohumol according to Bartmanska and co-workers. The
spectral characteristic of the obtained substance was in agreement
with literature data.22
(C-300), 30.8 (C-b), 46.9 (C- ), 56.5 (C60-OCH3), 71.5 (C-2000), 86.5 (C-
a
4,20,40-Trihydroxy-60-methoxy-3-prenyl-
a
,b-dihydrochalcone
50), 92.7 (C-200), 106.3 (C-30), 106.4 (C-10), 116.0 (C-3, C-5), 130.2
(C-2, C-6), 133.3 (C-1), 156.5 (C-4), 162.6 (C-60), 165.1 (C-40), 168.2
(C-20), and 205.6 (C@O); IR (film cmꢁ1): 3057 (m), 1724 (s), 1507
(m), 1458 (m), 1420 (m), 1363 (s), 1222 (s), and 1091 (m); HR ESI-
MS m/z: 371,1421 [MꢁH]ꢁ (calcd for C21H24O6 ꢁ H, 371.1489);
(4) was isolated from the Fusarium tricinctum AM16 culture after a
14-day transformation of xanthohumol (1) (29 mg, yield 24%). 1H
NMR (600 MHz, CD3OD) d: 1.64 (3H, s, H-500), 1.75 (3H, s, H-400),
2.83 (2H, m, H-b), 3.20 (2H, m, H-a
, H-100), 3.20 (2H, m, H-100, H-
a
), 3.82 (3H, s, C60-OCH3), 5.18 (1H, m, H-200), 5.98 (1H, s, H-50),
½
a 2D0
+32.453 (c 0.145, MeOH); UV (MeOH) kmax 295.1 nm.
ꢀ
6.70 (2H, m, H-3, H-5), and 7.03 (2H, m, H-2, H-6); 13C NMR
Xanthohumol H (30-[300-hydroxy-300-methylbutyl]-4,20,40-trihy-
(151 MHz, CD3OD) d: 17.9 (C-400), 22.2 (C-100), 26.0 (C-500), 31.6 (C-
droxy-60-methoxychalcone) (7) was isolated from Penicillium albi-
dum AM79 culture after a 8-day transformation of xanthohumol
(1) (36.0 mg, yield 22.84%). 1H NMR (600 MHz, acetone-d6) d:
b), 47.5 (C-
a
), 55.9 (C60-OCH3), 91.2 (C-50), 105.8 (C-10), 109.2 (C-
30), 116.2 (C-3, C-5), 124.2 (C-200), 130.3 (C-2, C-6), 131.3 (C-300),
Please cite this article in press as: Tronina, T.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.01.026

T. Tronina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
1.13 (6H, s, H-1000, H-3000), 1.46 (2H, m, H-200), 2.50 (2H, m, H-100),
3.86 (3H, s, C60-OCH3), 6.08 (1H, s, H-50), 6.84 (2H, m, H-3, H-5),
7.57 (2H, m, H-2, H-6) 7.67 (1H, d, J = 15,5 Hz, H-b), 7.76 (1H, d,
Microorganisms were maintained on agar slants and grown on a
Sabouraud medium (fungi) or nutrient broth (bacteria).
J = 15.5 Hz, H-
a
), and 14.60 (C20-OH); 13C NMR (151 MHz, ace-
2.3. Conditions for biotransformations
tone-d6) d: 17.2 (C-100), 29.0 (C-400, C-500), 42.6 (C-200), 55.7 (C60-
OCH3), 69.0 (C-300), 90.9 (C-50), 104.6 (C-30), 108.6 (C 10), 116.0
The cultures were shaken at 25 °C in 100-ml Erlenmeyer flasks
with 30 ml of the medium in the screening studies and in 300-ml
Erlenmeyer flasks with 100 ml of the medium in the preparative-
scale transformation.
In the screening tests a solution of xanthohumol (1) (10 mg in
0.5 ml of methanol) was added to 2-day bacterial or 3-day fungal
cultures. After 7 days of incubation on a rotary shaker (25 °C, in
the darkness) the metabolites and any remaining substrate were
extracted with ethyl acetate.
In the preparative biotransformations 120 mg of xanthohumol
(1) dissolved in 8 ml of methanol was equally distributed among
four flasks with 3-day fungal cultures (2 ml each). The reactions
were carried out under the same conditions as in screening tests
and continued until the substrate was metabolized (the progress
of conversion was monitored by HPLC). All the experiments were
performed in duplicate. Substrate control consisted of xanthohu-
(C-3, C-5), 123.9 (C-1), 126.0 (C-a), 130.5 (C-2, C-6), 142.4 (C-b),
159.9 (C-4), 160.3 (C-60), 162.5 (C-40), 164.7 (C-20), and 191.7
(C@O); HR ESI-MS m/z: 371.1457 [MꢁH]ꢁ (calcd for C21H24O6 ꢁ H,
371.1489); UV (MeOH) kmax: 366.9 nm.
200-(2000-Hydroxyisopropyl)-dihydrofurano[400,500:30,40]-4,20-dihy-
droxy-60-methoxychalcone (5), mixture of diastereoisomers of
(2S,200S) and (2S,200R) 200-(2000-hydroxyisopropyl)-dihydrofurano-
[400,500:7,8]-40-hydroxy-5-methoxyflavanone (8) and (Z)-200-(2000-
hydroxyisopropyl)-dihydrofurano-[400,500-6,7]-30,40-dihydroxy-4-
methoxyaurone (9) were obtained by transformation of xanthohu-
mol in Aspergillus ochraceus AM465 culture, which was described
in our earlier work.25
2.2. Microorganisms
Seventy eight microorganisms (fungi and bacteria) used for
screening were purchased from the Department of Pharmaceutical
Biology and Botany of the of Wrocław Medical University, Poland
(indexed AM), Department of Forest Pathology of the Agricultural
University of Kraków, Poland (indexed ARK), Department of Chem-
istry of the Wrocław University of Environmental and Life Sciences,
Poland (indexed KCh), Department of Biotechnology and Food
Microbiology of the Wrocław University of Environmental and Life
Sciences, Poland (indexed AR), Department of Plant Protection of
Wrocław University of Environmental and Life Sciences, Poland
(indexed UPF), Polish Collection of Microorganisms (indexed
PCM) and American Type Culture Collection (indexed ATCC). The
cultures used for preliminary screening were as follows: Absidia
glauca AM254, Acinetobacter lwoffii PCM2235, Acremoniella atra
AM29, Alternaria longipes AM266, Armillaria mellea AM296, Ascos-
phaera apis AM496, Botrytis cinerea AM235, Candida tropicalis
PCM2681, Cenangium ferruginosum ARK56, Chaetomium indicum
AM32, C. indicum AM158, Cladosporium avellaneum AM135, Crume-
nulopsis sororia ARK59, Epithyrium resinae AM51, Fusarium avenace-
um AM11, F. avenaceum AM12, Fusarium culmorum AM7, F.
culmorum AM9, F. culmorum AM10, F. culmorum AM196, F. culmo-
rum AM282, Fusarium equiseti AM22, Fusarium oxysporum AM13,
F. oxysporum AM21, F. oxysporum UPF727, Fusarium scirpi AM199,
Fusarium semitectum AM20, Fusarium solani AM203, Fusarium tri-
cinctum AM16, Fusicoccum amygdali AM258, Gloeophyllum odora-
tum ARK58, Inonotus radiatus ARK70, Laetiporus sulphurens
AM498, Micrococcus luteus PCM525, Mortierella vinacea AM149,
Mortierella isabellina AM212, Mucor circinelloides AM385, Malachius
spinosus AM398, Nigrospora oryzae AM8, Nocardia sp. KCh1, Penicil-
lium albidum AM79, Penicillium camembertii AM83, Penicillium cher-
mesinum AM113, Penicillium chrysogenum AM112, Penicillium
citrinum AM354, Penicillium diversum AM388, Penicillium frequen-
tans AM351, P. frequentans AM359, Penicillium lilacinum AM111,
Penicillium purpurogenum AM80, Penicillium spinulosum AM114,
Parakeijia thomi AM91, Penicillium urticae AM84, Penicillium vermi-
culatum AM30, P. vermiculatum AM81, Penicillium vinaceum
AM110, Pholiota aurivella AM521, P. aurivella AM522, Piptoporus
betulinus ARK39, Penicillium betulinus ARK57, P. betulinus AM475,
Papularia rosea AM17, Poria placenta AR37, Penicillium placenta
AR38, Poria vaporaria AM270, Pseudomonas fragii PCM1856,
Pseudomonas fluorescens PCM2123, Pseudomonas putida PCM2124,
P. putida PCM2153, Pycnidiella resinae ARK50, Rhodotorula glutinis
AM242, Rhodotorula rubra AM4, Spicaria fusispora AM136, Syrnola
violacea AM439, Sclerophoma pythiophila ARK55 and Yarrowia
lipolytica AR71.
mol and
microorganisms.
a
sterile growth medium incubated without
2.4. Reaction work-up and product analysis
The fermentation media were extracted twice with 25 ml of
ethyl acetate. The extracts were evaporated, and the residue dis-
solved in methanol and analyzed by TLC and HPLC. TLC was carried
out on Merck silica gel 60, F254 (02 mm thick) plates. HPLC was per-
formed on a Waters 2695 Aliance instrument with a photodiode
array detector Waters 2996 (detection at 280 and 370 nm wave-
length) using the analytical HPLC column Agilent ZORBAX Eclipse
XDB 5
lm (46 ꢂ 250 mm) at the flow rate of 1 ml/min A linear sol-
vent gradient from 40% to 60% aq MeCN containing 1% HCOOH over
40 min was used. The products of xanthohumol biotransformation
were separated by column chromatography on silica gel 60 (230–
400 mesh, Merck) using chloroform/methanol (9:1 v/v) as eluent.
Quantitative analysis of the ethyl acetate extract of the biotransfor-
mation mixture was performed by means of HPLC, using the cali-
bration curves for the substrate and for all the isolated products
NMR spectra (1H NMR, 13C NMR, DEPT 135°, 1H–1H NMR (COSY),
1H–13C NMR (HMQC)) were recorded on a MHz DRX Bruker
Avance™ 600 (600 MHz) instrument in DMSO-d6, acetone-d6, or
in CD3OD. UV spectra were run on a Spectrofotometer Cintra
303, GBC, in methanol. Optical rotations were measured on an
Autopol IV Rudolph polarimeter in methanol and CD measure-
ments on a JASCO J-715/ORD spectropolarimeter in methanol. Po-
sitive and negative-ion ESI-MS spectra were taken on a Bruker
micrOTOF-Q spectrometer IR spectra were recorded on a Ther-
mo-Nicolet IR 300 FTIR spectrometer.
2.5. Analysis
2.5.1. Free radical-scavenging activity assay
Free radical-scavenging activity was measured essentially by
the method proposed by Sharma and Bhat26 using 1,1-diphenyl-
2-picrylhydrazyl (DPPH). Briefly, 10 ml of 0.5 mM methanolic solu-
tion of DPPH radical was added to 2 ml of 0.1 M acetate buffer (pH
5.5) and 2 ml of methanolic test samples of different concentra-
tions. Final concentration of DPPH in each sample was 100
lM
(0.5 mol). The solvents and other chemicals were of analytical
l
grade. The mixture was shaken vigorously and left to stand for
30 min at room temperature in the dark. The absorbance at
517 nm by DPPH was measured by UV–vis spectrophotometry
L-ascorbic acid was used as a positive control. The percentage of
Please cite this article in press as: Tronina, T.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.01.026

4
T. Tronina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
inhibition of the DPPH radical by the samples was calculated as
follows:
3. Results and discussion
3.1. Biotransformation results
Inhibition ½%ꢀ ¼ 100 ꢂ ðABSt¼0 ꢁ ABSt¼30Þ=ABSt¼0
where ABSt = 0 was the absorbance of DPPH at time 0 and ABSt = 30
was the absorbance of DPPH after 30 min of incubation. Sample
concentration providing 50% of inhibition (IC50) was calculated
from the graph plotting inhibition percentage against compound
concentration in sample. All measurements were made in tripli-
cates. One-way analysis of variance (ANOVA) was performed with
SPSS. The differences were considered significant at p <0.05 using
the post hoc evaluation with Fisher0s least significant difference
test.
A few biotransformation studies have been reported directly on
chalcones, which was summarized by Das and Rosazza.28 Some of
them concern transformations of xanthohumol (1) by fungi,25,29–31
human and rat liver microsomes32,33 and rats.34 In the light of our
interest25,31 in the microbiological xanthohumol transformation,
we have endeavored to search for strains outside genus Beauveria
and Aspergillus that would offer new route to novel biologically ac-
tive chalcone metabolites. Seventy eight microorganisms were
screened for potential xanthohumol transformation activity. Sev-
eral of them showed the capability of transforming xanthohumol.
Fungi Fusarium tricinctum AM16, F. avenaceum AM11, F. oxysporum
UPF727, Spicaria fusispora AM136 and Penicillium albidum AM79
were selected for scale-up studies, because they gave the products
with the highest efficiency compared to the other microorganisms.
Preparative-scale biotransformations afforded four metabolites:
two chalcones (5, 7) and two dihydrochalcones (4, 6). One of them,
200-(2000-hydroxyisopropyl)-dihydrofurano-[400,500:30,40]-40,2-dihy-
2.5.2. Antiproliferative assay in vitro
2.5.2.1. Cells.
The following established in vitro cancer cell
lines were applied: MCF-7 (human breast carcinoma), PC-3 (hu-
man prostate cancer) and HT-29 (human colon cancer). All cancer
cell lines were obtained from American Type Culture Collection
(Rockville, Maryland, USA) and are being maintained in the Insti-
tute of Immunology and Experimental Therapy (Wrocław, Poland).
MCF-7 cells were cultured in Eagle medium supplemented with
droxy-60-methoxy-
a,b-dihydrochalcone (6) is a new compound.
2 mM L-glutamine and 10 mM sodium pyruvate, 10% fetal bovine
Although cyclisation of prenyl group to dihydrofurane deriva-
tives is rather common biotransformation pathway of prenylated
flavonoids,25,29,32,34 there is extremely little information concern-
serum and 0.8 mg/L of insulin (all from Sigma–Aldrich Chemie
GmbH, Steinheim, Germany). PC-3 and HT-29 cells were cultured
in RPMI 1640+Opti-MEM (1:1) (both from Gibco, Scotland, UK),
ing reduction of
a,b-double bond in natural, multifunctionalized
supplemented with 2 mM L-glutamine, 5% fetal bovine serum (all
chalcones. There are reports related to conversion of chalcones to
dihydrochalcones by fungi Aspergillus flavus,35 Saccharomyces cere-
visiae in biphasic system36 and by plant cell culture of Morus ni-
gra.37 Dihydrochalcones were described also as products of the
C-ring cleavage in flavanones by species of Aspergillus, Penicillium
and Streptomyces.28,38
from Sigma–Aldrich Chemie GmbH, Steinheim, Germany). HT-29
cells medium was additionally supplemented with 10 mM sodium
pyruvate. All culture media were also supplemented with antibiot-
ics: 100 units/ml penicillin, and 100 lg/ml streptomycin (both
from Polfa Tarchomin SA, Warsaw, Poland). All cell lines were
grown at 37 °C with 5% CO2 humidified atmosphere.
Twenty-four hours before addition of tested agents, the cells
were plated in 96-well plates (Sarstedt, Germany) at a density
3.1.1. Transformation by Fusarium tricinctum AM16
It was concluded that the fungus reduced
a,b-double bond of
of 104 cells per well in 100
l
l of culture medium. An assay
was performed after 72 h of exposure to varying concentrations
of the tested agents (from 0.1 to 100 g/ml). The results were
the substrate. Molecular ion peak at m/z 357.1755 [M+H]+ in the
high-resolution electrospray ionization mass spectral data (HR-
ESI-MS) corresponded to the C21H24O5 elemental formula. The
structure of the resulting product—4,20,40-trihydroxy-60-methoxy-
l
calculated as the IC50 (inhibitory concentration 50%), the concen-
tration of tested agent which inhibits 50% of the proliferation of
the cancer cell population. IC50 values were calculated for each
experiment separately and mean values SD are presented in
the table. Each compound at each concentration was tested in
3-prenyl-a,b-dihydrochalcone (4)—was easily determined by com-
parison of its spectroscopic data with that of the starting material
1.25 The 1H NMR spectrum showed no characteristic chalcone ole-
finic proton signals. New signals at dH 2.83 ppm (2H, m) and
3.20 ppm (2H, m) were observed, which suggested reduction of
triplicate in
a single experiment, which was repeated 3–5
a
,b-double bond. This was further supported by the 13C NMR of 4
times. Ethanol, which was used as a solvent (in a dilution corre-
sponding to its highest concentration applied to the tested com-
pounds), did not exert any inhibitory effect on cell proliferation.
To evaluate antiproliferative effect of tested compounds, the SRB
method was applied.
which showed significant upfield shifts of
a-carbon (D 76.3 ppm)
and b-carbon (
D 110.9 ppm) signal, that were consistent with ref-
erence shift values.39 The protons connected to them were ascribed
to dH 2.83 and 3.20 ppm by HMQC experiment. DEPT spectrum
confirmed that signals observed at 31.6 and 47.5 ppm (C-b and
2.5.2.2. SRB assay.
The details of this technique were de-
C-a) derived from secondary carbon atoms. a,b-Dihydroxanthohu-
scribed by Skehan et al.27 The cells attached to the plastic were
fixed by gently layering cold 50% TCA on the top of the culture
medium in each well. The plates were incubated at 4 °C for 1 h
and then washed five times with tap water. The background optical
density was measured in the wells filled with culture medium,
without the cells. The cellular material fixed with TCA was stained
with 0.4% sulforhodamine B dissolved in 1% acetic acid for 30 min.
Unbound dye was removed by rinsing (four times) with 1% acetic
acid. The protein-bound dye was extracted with 10 mM unbuffered
Tris base for determination of optical density (at 540 nm) in a com-
puter-interfaced, 96-well microtiter plate reader Multiskan RC
photometer. One-way analysis of variance (ANOVA) followed by
a Mann–Whitney U Test was applied p-values <0.05 were consid-
ered significant.
mol (4) occurs naturally in hop as a minor component39 and there-
fore biotransformation of easily accessible xanthohumol (1) by
Fusarium tricinctum culture is a simple way to receive this active
compound, which also retains the status of ‘natural compound’,
in a reasonable amount.
3.1.2. Transformation by Spicaria fusispora AM136
After 9 days of transformation of 120 mg of xanthohumol (1) 200-
(2000-hydroxyisopropyl)-dihydrofurano-[400,500:30,40]-4,20-dihy-
droxy-60-methoxychalcone (5) was isolated (21.32 mg, yield 17%).
Spectral data of this metabolite are in accordance with our previ-
ous paper.25 This product was formed also in biotransformation
of xanthohumol (1) by fungi Pichia membranifaciens,29 Aspergillus
ochraceus,25 human and rat liver microsomes32,33 and by rats.34
Please cite this article in press as: Tronina, T.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.01.026

T. Tronina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
Table 1
The process of transformation by Spicaria fusispora described here
DPPH radical scavenging ability of hop flavonoids (1–3)
and xanthohumol biotransformation products (4–9)
is the most efficient of them.
a
Antioxidant
IC50
3.1.3. Transformation by Fusarium avenaceum AM11 and
Fusarium oxysporum UPF727
Incubation of xanthohumol (1) both with Fusarium avenaceum
AM11 and Fusarium oxysporum AM727 gave single metabolite 6,
which turned out to be the product of the prenyl group cyclisation
1
2
3
4
5
6
7
8
9
1.98 0.24
2.37 0.40
35.42 0.11
12.97 0.77
2.24 0.15b
20.44 0.59
3.34 0.26
87.37 7.22b
0.23 0.03b
0.52 0.25
and a,b-double bond reduction. In comparison to the spectrum of
substrate (1), some characteristic changes, described above for
metabolites 4 (reduction) and 5 (cyclisation), were observed. This
product had new resonances at dH 2.85 ppm (H-b) and dH
L-Ascorbic acid (reference)
3.24 ppm (H-a), which suggested hydrogenation of
a,b-double
a
bond. The upfield shifts of C-
a
(D
76.9 ppm) and C-b (D
Concentration
inhibition.
lMantiox
/
l
MDPPH for
a
50%
111.7 ppm) assigned in DEPT experiment to secondary carbon
atoms, supported the postulated reduction. Cyclisation of the pre-
nyl group to dihydrofurane derivative was confirmed by the pres-
ence of three dublets of dublets at dH 3.01 ppm (1H, dd, J = 15.1,
9.6 Hz, H-300a), 3.07 ppm (2H, dd, J = 15.1, 7.9 Hz, H-300b) and
4.75 ppm (1H, dd, J = 9.6, 7.9 Hz, H-200). High-resolution electro-
spray ionization mass spectral data (HR-ESI-MS) of compound 6
showed the molecular ion [MꢁH]ꢁ at m/z 371.1421 which estab-
lished the molecular formula as C21H24O6 (calcd for C21H24O6 ꢁ H,
371.1489). All these results fully confirmed the structure of metab-
olite 6 as 200-(2000-hydroxyisopropyl)-dihydrofurano-[400,500:30,40]-
b
Results presented in our previous paper (Tronina
et al. 24).
weaker antioxidant, however flavanone with cyclised prenyl group
(8) was 44-fold weaker antioxidant. Reduction of ,b-unsaturated
a
bond occurring in dihydrochalcones 4 and 6, resulted in decrease
of the activity. No significant differences in DPPH radical scaveng-
ing activities were found between prenylchalcone 1 and the chal-
cones with modified prenyl group: 2, 5 and 7. A 30,40-catechol
structure in the B-ring strongly enhances DPPH radical scavenging
activity of aurone 9. This arrangement is a salient feature of the
most potent peroxyl, superoxide and peroxynitrite radicals scav-
engers.42,43 Aurone 9, showed the most potent activity with the
40,2-dihydroxy-60-methoxy-
a,b-dihydrochalcone. To the best of
our knowledge this metabolite have not been previously reported
in the literature.
IC50 value 0.23 lM/lMDPPH, and was 8.6-fold stronger antioxidant
3.1.4. Transformation by Penicillium albidum AM79
than xanthohumol (1) and 2.3-fold than L-ascorbic acid.
Penicillium albidum AM79 oxidized xanthohumol (1) at the dou-
ble bond of prenyl group to xanthohumol H (7). This metabolite
showed the [MꢁH]ꢁ peak at m/z 371.1457 (calcd for C21H24O6 ꢁ H,
371.1489), which established the molecular formula as C21H24O6.
The UV spectrum showed maximum absorption band at
366.9 nm, which indicated that it was a chalcone derivative. Its
1H NMR spectrum differed from that of xanthohumol (1)25 in
two important respects. Firstly, olefinic proton signal at C-200
(5.14 ppm, 1H, m) was replaced by a new signal at dH 1.46 ppm
(2H, m). Secondly, proton signals of prenyl methyl groups observed
as two singlets at 1.69 ppm (H-4000, 3H, s) and 1.60 ppm (H-5000, 3H,
s) in the substrate spectrum were replaced by one singlet of six
protons at 1.13 ppm. Additionally, an upfield shift of the H-100 res-
onance signal (dH 2.50) was observed, which suggested introduc-
tion of a hydroxyl group at C-300. The proposed structure was
further confirmed by 13C NMR, in which two olefinic carbon signals
at dC 123.0 and 130.5 ppm were replaced by two signals at dC 42.6
and 69.0 ppm. A high value of C-300chemical shift (69.0 ppm) and
the same values of chemical shifts of C-400 and C-500 signals
(29.0 ppm) indicate the presence of a hydroxyl group at C-300 posi-
tion. The NMR data of this compound are in agreement with those
reported in the literature.40
3.3. Antiproliferative activity
A screening test using human cancer cell lines is usually per-
formed as an initial step in the evaluation of chemicals for cancer
prevention and treatment.27 Because some hop prenylflavonoids
are known for their anticancer activity,14–20 we have decided to
investigate the activity of some minor constituents of hops that
have never been tested so far, and also their derivatives obtained
by means of biotransformation. We have chosen three human can-
cer cell lines: breast cancer (MCF-7), colon cancer (HT-29) and
prostate cancer (PC-3), which were earlier used to examine the
antiproliferative activity of hop flavonoids, among them xanthohu-
mol (1) and isoxanthohumol (3).14,18
The in vitro cytotoxic activity was evaluated using SRB assay
and expressed as IC50, the concentration of the compound that
inhibited proliferation rate of tumor cells by 50% as compared to
the untreated control cells. Cisplatin was applied as a reference
cytotoxic agent (positive test control). The results of the screening
are summarized in Table 2. Human breast cancer cell line MCF-7
appears to be most sensitive to the cytotoxic effects of compounds
1–9, compared to the two other cancer cell lines. It is important to
note that compounds 1 and 4 exhibited the cytotoxic activity high-
3.2. Antioxidant activity
er than cisplatin. Reduction of the a,b-double bond increased anti-
proliferative properties (4 vs 1), while prenyl moiety modification
resulted in decrease of these properties (1 vs 2, 5, 7 and 3 vs 8).
Isomerisation of chalcone to flavanone led also to decrease in cyto-
toxic activity against MCF-7 cancer cells (1 vs 3). The last two
observations were similar to those described by Miranda et al.14
The best antioxidant among the tested compounds appeared aur-
one 9, however, it was less active against MCF-7 cancer cells than
the substrate 1.
Most of the beneficial health effects of flavonoids are attributed
to their antioxidant properties. With this in mind, both hop pren-
ylflavonoids1,2,41 and microbial metabolites of xanthohumol,3,25
were in the first approach tested for their antioxidant activity.
We assessed the abilities of a range of structurally diverse flavo-
noids to scavenge DPPH radicals, which is rapid, simple and eco-
nomic method to determine antioxidant activity of phenolic
compouds. Table 1 showed that the greatest DPPH radical scaveng-
ing activity exhibited chalcones 1, 2, 5, 7 and aurone 9. The chal-
cone–flavanone cyclisation results in decrease of the activity.
Compared to xanthohumol (1), isoxanthohumol (3) was 18-fold
Comparison of the sensitivity of PC-3 and MCF-7 cancer
cell lines indicated that, except for reduction of
a,b-double bond,
all the modifications of xanthohumol molecule led to the distinc-
tive reduction in activity. Xanthohumol (1) showed stronger
Please cite this article in press as: Tronina, T.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.01.026

6
T. Tronina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 2
Supplementary data
In vitro antiproliferative activity of hop flavonoids (1–3) and xanthohumol biotrans-
formation products (4–9) against human cancer cell lines
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.026.
a
Compound
Cancer cell line, IC50
PC-3
(l
M)
MCF-7
HT-29
References and notes
1
2
3
4
5
6
7
8
10.95 1.03
20.60 0.22
26.54 12.68
9.15 0.62
49.56 5.45
61.82 18.23
21.81 6.98
105.64 13.31
48.31 13.89
11.55 0.48
10.67 1.06
37.88 13.90
91.31 8.92
78.83 8.83
88.82 4.15
74.41 23.44
90.52 3.46
85.69 3.47
122.31 38.05
90.52 3.46
114.13 9.65
18.96 2.08
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´
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Acknowledgment
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This research was supported financially by the European Union
within the European Regional Development Found (Grant No. POIG
01.03.01-00-158/09).
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Please cite this article in press as: Tronina, T.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.01.026