Microbial Transformations of 7-Methoxyflavanone

Article abstract of DOI:10.3390/molecules171214810

Microbial transformations of racemic 7-methoxyflavanone using strains of the genus Aspergillus (A. niger KB, A. ochraceus 456) and the strain Penicillium chermesinum 113 were described. The strain A. niger KB catalysed carbonyl group reduction, leading to (±)-2,4-cis-7-methoxyflavan-4-ol. Biotransformation with the help of A. ochraceus 456 gave two products: (+)-2,4-trans-7-methoxyflavan-4-ol and 4'-hydroxy-7-methoxyflavone. Transformation by means of P. chermesinum 113 resulted in a dihydrochalcone product, 4,2'-dihydroxy-4'-methoxydihydrochalcone. DPPH scavenging activity test proved that all the biotransformations products have higher antioxidant activity that the substrate.

Full text of DOI:10.3390/molecules171214810

Molecules 2012, 17, 14810-14820; doi:10.3390/molecules171214810
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Microbial Transformations of 7-Methoxyflavanone
Edyta Kostrzewa-Susłow * and Tomasz Janeczko
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25,
Wrocław 50-375, Poland; E-Mail: janeczko13@interia.pl
* Author to whom correspondence should be addressed; E-Mail: ekostrzew@gmail.com;
Tel./Fax: +48-713-284-124.
Received: 2 November 2012; in revised form: 16 November 2012 / Accepted: 30 November 2012 /
Published: 11 December 2012
Abstract: Microbial transformations of racemic 7-methoxyflavanone using strains of the
genus Aspergillus (A. niger KB, A. ochraceus 456) and the strain Penicillium chermesinum
113 were described. The strain A. niger KB catalysed carbonyl group reduction, leading to
(±)-2,4-cis-7-methoxyflavan-4-ol. Biotransformation with the help of A. ochraceus 456
gave two products: (+)-2,4-trans-7-methoxyflavan-4-ol and 4'-hydroxy-7-methoxyflavone.
Transformation by means of P. chermesinum 113 resulted in a dihydrochalcone product,
4,2'-dihydroxy-4'-methoxydihydrochalcone. DPPH scavenging activity test proved that all
the biotransformations products have higher antioxidant activity that the substrate.
Keywords: biotransformation; 7-methoxyflavanone; Aspergillus niger; Aspergillus ochraceus;
Penicillium chermesinum
1. Introduction
Flavonoids are polyphenolic compounds with diverse chemical structures, which are widely found
in plants. Apart from plants, a natural capability to carry out biosynthesis of flavonoid compounds is a
feature of some endophytic fungi [1]. Animal and human organisms do non synthesize flavonoids [2,3].
Their specific properties make them useful for pharmaceutical, cosmetics and food industry. Therefore,
there is growing interest in chemical synthesis of flavonoids, as well as in their biotechnological
production [4–9]. The therapeutic potential and low toxicity of flavonoids are accompanied with
relatively little information about their metabolic pathways in living organisms. That is why the
attention has been directed to biocatalysis [10].

Molecules 2012, 17
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The microorganisms used in biotransformation of flavonoid compounds have enzymatic systems
capable of performing various chemical reactions, including reduction, hydroxylation, O-methylation
and hydrolysis [11,12]. Analytical tests carried out during the course of biotransformation allow
tracing of the metabolic transformations of flavonoids [13–15]. The products are often new
compounds, not described in the literature so far and difficult to obtain by chemical synthesis.
Moreover, they have often high antioxidant properties [16,17].
Biotransformation of compounds with a methoxy group in the C-7 position of flavanone were
described by Ibrahim and co-workers [18]. The strain Cunninghamella elegans NRRL 1392
transformed 7-O-methylnaringenin (sakuranetin) into naringenin and naringenin-4'-sulfate, and
5,3',4'-trihydroxy-7-methoxyflavanone into eriodictyol-4'-sulfate, whereas biotransformation of
5,4'-dihydroxy-7,3'-dimethoxyflavanone afforded homoeriodictyol (5,7,4'-trihydroxy-3'-methoxyflavanone)
and homoeriodictyol-7-sulfate. The observed sulfatation and O-demethylation proceeded
regio-selectively [18].
The objective of our research was to transform racemic 7-methoxyflavanone into optically pure
products with higher antioxidant properties than the starting substrate.
2. Results and Discussion
An initial screening of 27 filamentous fungi of the genera Aspergillus, Penicillium, Coryneum,
Nectria, Chaetomium, Absidia, Spicaria, and Cryptosporiopsis [19] allowed us to select three
microorganisms (Aspergillus niger KB, Aspergillus ochraceus 456 and Penicillium chermesinum 113)
that were capable of transforming racemic 7-methoxyflavanone (1).
The wild strain A. niger KB, as in the case of flavanone [20], 6-hydroxyflavanone [20] and
7-hydroxyflavanone [19], performed a reduction of the carbonyl group of 7-methoxyflavanone (1)
(Scheme 1).
Scheme 1. Reduction of the carbonyl group of 7-methoxyflavanone with A. niger KB.
3'
4'
2'
B
8
5'
O
C
O
CH3O
7
2
CH3O
Aspergillus
6'
A
niger KB
3
6
4
5
O
OH
(±)-7-methoxyflavanone (1)
(±)-2,4-cis-7-methoxyflavan-4-ol (2)
Racemic 7-methoxyflavan-4-ol (2) was obtained in 76% yield (76 mg from 100 mg of the substrate)
after 9 days of biotransformation. The reaction was continued until the substrate was fully consumed.
1
The reduction of the carbonyl group of 7-methoxyflavanone (1) was confirmed in the H-NMR
spectrum of 2 by the presence of a wide singlet of one proton at  = 5.05 ppm, corresponding to H-4.
In the 13C-NMR the chemical shift of C-4 is moved from  = 192.2 ppm for substrate 1 to  = 65.6 ppm for
7-methoxyflavan-4-ol (2). Additionally, in the IR spectrum of 2 a new hydroxyl group absorption band

Molecules 2012, 17
14812
1
appeared at 3225 cm−1. In the H-NMR spectrum of 2 the chemical shifts of H-2, H-4, H-3ax, and
H-3eq, as well as the respective coupling constants were compared with the carbonyl group reduction
products obtained in transformation of 6- and 7-hydroxyflavanone using the strain A. niger KB (flavan-4-
ol [20], 6-hydroxyflavan-4-ol [20], and 7-hydroxyflavan-4-ol [19]) (Table 1). This comparison
indicates 2,4-cis configuration of 7-methoxyflavan-4-ol (2). The proof of this configuration with the
help of X-ray analysis was given in our earlier works [19,20]. Observed values of the coupling
constants: J2,3eq = 2.0 Hz and J2,3ax = 11.4 Hz show that the phenyl group is in a pseudoequatorial
orientation, whereas J4,3eq = 6.3 Hz and J4,3ax = 10.2 Hz confirm a pseudoequatorial position of the
hydroxyl group at C-4. Protons H-2 and H-4 are in pseudoaxial positions.
1
Table 1. Selected H-NMR data for 2,4-cis-flavan-4-ol [20], 2,4-cis-7-hydroxyflavan-4-ol
[19], 2,4-cis-6-hydroxyflavan-4-ol [20] and 2,4-cis-7-methoxyflavan-4-ol (2).
Compound
2,4-cis-Flavan-4-ol
2,4-cis-7-
 H-2
5.17
 H-4  H-3ax  H-3eq J3ax-3eq J2-3ax J 2-3eq J 4-3ax J4-3eq
5.08
2.13
2.51
13.1
11.6
1.8
10.6
6.3
5.15
5.09
5.17
5.00
2.09
2.49
13.2
11.4
1.8
9.7
6.2
Hydroxyflavan- 4-ol
2,4-cis-6-
4.98
5.05
1.98
2.13
2.35
2.52
12.9
13.2
11.9
11.4
1.9
2.0
10.8
10.2
6.4
6.3
Hydroxyflavan-4-ol
2,4-cis-7-
Methoxyflavan-4-ol
The enzymatic system of A. ochraceus 456 catalysed transformation of (±)-7-methoxyflavanone (1)
into two products: (+)-2,4-trans-7-methoxyflavan-4-ol (3) and 4'-hydroxy-7-methoxyflavone (4)
(Scheme 2). After 9 days of biotransformation (+)-2,4-trans-7-methoxyflavan-4-ol (3) (4.9% yield,
4.9 mg/100 mg of the substrate), and 4'-hydroxy-7-methoxyflavone (4) (2.3% yield, 2.3 mg/100 mg of
the substrate) were isolated from the reaction mixture.
Scheme 2. A. ochraceus 456 catalysed transformation of (±)-7-methoxyflavanone (1).
O
CH3O
OH
(+)-2,4-trans-7-methoxyflavan-4-ol (3)
O
CH3O
Aspergillus
ochraceus 456
OH
CH3O
O
O
O
(1)
(±)-7-methoxyflavanone (1)
4'-hydroxy-7-methoxyflavone (4)

Molecules 2012, 17
14813
When monitoring the reaction progress by means of TLC and HPLC we noticed that the amount of
product 4, which is visible starting from the third day of the reaction, was increasing slightly. The
reduction product: (+)-2,4-trans-7-methoxyflavan-4-ol (3) appears in the sixth day, however in the
ninth day of the process its amount is higher than the amount of the independently formed 4'-hydroxy-
7-methoxyflavone (4) (Table 2).
Table 2. Biotransformation of 7-methoxyflavanone (1), yield (%) of products: 2,4-trans-7-
methoxyflavan-4-ol (3) and 4'-hydroxy-7-methoxyflavone (4), according to HPLC
(screening tests).
Biotransformation products (%)
Unreacted substrate
Time of Incubation
Microorganism
(days)
4
3
0
(%)
82.0
35.7
18.7
9.1
1
3
6
9
0
2.1
2.3
2.5
0
Aspergillus
ochraceus 456
3.7
5.3
From the first day of the reaction we have observed a considerable decrease in the amount of the
substrate 1, which was not proportional to the amount of products formed (Table 2). These, along with
low yield of products, indicate that both 7-methoxyflavanone (1) and products 3 and 4 may undergo
degradation in the culture of A. ochraceus 456.
1
In the H-NMR spectrum of 3 we can see a multiplet at  = 5.07 ppm, integrating for one proton,
which does not occur in the spectrum of substrate 1 and which corresponds to H-4. Whereas, in the
13C-NMR there is a change in the chemical shift of C-4 from  = 192.2 ppm for 7-methoxyflavanone
(1) to  = 65.6 ppm for 2,4-trans-7-methoxyflavan-4-ol (3), which is typical for the reaction of
reduction. The configuration of 2,4-trans-7-methoxyflavan-4-ol (3) was confirmed by comparing the
1H-NMR spectrum of product 3 with the described earlier spectrum of another carbonyl group
reduction product—2,4-cis-7-methoxyflavan-4-ol (2) (Table 3).
Table 3. Selected 1H-NMR data for (±)-2,4-cis-7-methoxyflavan-4-ol (2) and
(+)-2,4-trans-7-methoxyflavan-4-ol (3).
Compound
(±)-2,4-cis-7-Methoxy-
flavan-4-ol (2)
 H-2
 H-4
 H-3ax
 H-3eq
J3ax-3eq
J2-3ax
J2-3eq
J4-3ax
J4-3eq
5.17
5.05
2.13
2.52
13.2
11.4
2.0
10.2
6.3
(+)-2,4-trans-7-Methoxy-
flavan-4-ol (3)
5.17
5.07
2.95
2.52
13.2
11.5
1.8
3.2
6.2
The observed values of the coupling constants: J2,3ax = 11.5 Hz, J2,3eq = 1.8 Hz, J4,3ax = 3.2 Hz, and
J4,3eq = 6.2 Hz suggest a pseudoequatorial orientation of the phenyl group and a pseudoaxial position of
the hydroxyl group at C-4. Confirmation of the 2,4-trans configuration by means of X-ray analysis was
described in our earlier papers [19,20].
For the obtained 2,4-trans-7-methoxyflavan-4-ol (3) the measured specific optical rotation was
20
[α] = +2.57 (c = 0.7, CH3OH), and the enantiomeric excess (by HPLC, chiral column) ee = 30%. For
546

Molecules 2012, 17
14814
the unreacted substrate (1) isolated from the reaction mixture the data was as follows:
20
[α] = +6.28 (c = 2.1, CH3OH) and ee = 24%.
546
The structure of the independently formed 4'-hydroxy-7-methoxyflavone (4) was established by
1
means of H NMR and 13C-NMR. Hydroxylation at C 4' in ring B is proved by two doublets
integrating each for 2 H at  = 6.95 ppm (J = 8.6 Hz) and  = 7.81 ppm (J = 8.7 Hz), corresponding to
13
H-3', H-5' and H-2', H-6', respectively. In the C-NMR we observe a change in C-4' chemical shift
1
from  = 128.8 ppm for substrate (1) to  = 160.5 ppm for product 4. In the H-NMR spectrum of
7-methoxyflavone (1) the signal of H-2 appears at  = 5.47 ppm as a doublet of doublets (J = 13.3 Hz
and J = 2.8 Hz), the signal of H-3ax at  = 3.04 ppm is a doublet of doublets (J = 16.9 Hz and J = 13.3 Hz)
and the signal of H-3eq at  = 2.84 ppm is also a doublet of doublets (J = 16.9 Hz and J = 2.9 Hz). In
1
the H-NMR of 4'-hydroxy-7-methoxyflavone (4) a one proton singlet is visible at  = 5.69 ppm,
attributed to H-3, whereas the signal of H-2 disappears, which confirm the presence of a double bond
between C-2 and C-3 in ring C. Additionally, in the 13C-NMR the signals of C-2 and C-3 were moved
from  = 80.2 ppm and  = 44.3 ppm for the substrate (1) to  = 162.8 ppm and  = 104.0 ppm for the
dehydrogenation product 4.
Transformation of (±)-7-methoxyflavanone (1) by the strain P. chermesinum 113 led to formation of
product 5 of dihydrochalcone structure (Scheme 3).
Scheme 3. Transformation of (±)-7-methoxyflavanone (1) by the strain P. chermesinum 113.
3
3'
OH
4
4'
2
2'
3'
8
5
5'
2'
O
O
4'
CH3O
OH
O
2
CH3O
7
Penicillium

6
6'

6
chermesinum 113
3
5'
4
5
6'
(1)
4,2'-dihydroxy-4'-methoxydihydrochalcone (5)
(±)-7-methoxyflavanone (1)
4,2'-Dihydroxy-4'-methoxydihydrochalcone (5) was isolated after 10 days of biotransformation in
15% yield (15 mg/100 mg of the substrate). We expected a 4'-hydroxylation product with the C ring of
7-methoxyflavone intact, which would be analogous to the described earlier hydroxylation of
flavanone catalysed by the strain P. chermesinum 113 [15]. However, when monitoring the reaction
progress by TLC and HPLC we did not observe any products other than 4,2'-dihydroxy-4'-
methoxydihydrochalcone (5).
1
In the H-NMR spectrum of product 5 there are two triplets at  = 2.98 ppm and 3.19 ppm,
integrating for two protons each, corresponding to H-β and H-α and typical for a dihydrochalcone
structure. Opening of the ring C is additionally proved by the presence of the one proton singlet at
 = 12.6 ppm, which is attributed to 2'-OH. Substitution at C-4 is confirmed by two dublets at  = 6.77 ppm
and  = 7.11 ppm of identical coupling constants (J = 8.5 Hz) and integrating for 2 protons each. The
4-OH hydroxyl proton is visible as a singlet at  = 5.38 ppm.
In the UV spectra of the biotransformations products the strongest bathochromic shift in the
absorption maxima was observed for 4,2'-dihydroxy-4'-methoxydihydrochalcone (5): Δmax = 19 nm
(1st band), Δmax = 9 nm (2nd band), Δmax = 15 nm (3rd band) (Table 4).

Molecules 2012, 17
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Table 4. UV absorption of 7-methoxyflavanone (1) and its biotransformation products (2–5).
1st band
max [nm]
2nd band
max [nm]
3rd band
max [nm] log 
Compound

log 
4.18
3.89
3.81
4.37

log 
4.19
3.85
3.87
4.58
7-Methoxyflavanone (1)
2,4-cis-7-Methoxyflavan-4-ol (2)
2,4-trans-7-Methoxyflavan-4-ol (3)
4’-Hydroxy-7-methoxyflavone (4)
4,2’-Dihydroxy-4’-
235
229
234
234
273
282
281
276
310
-
3.89
-
-
-
309
4.29
254
4.24
282
4.43
325
4.13
methoxydihydrochalcone (5)
In the case of product 5 a considerable increase in molar absorption coefficient value () compared
to substrate 1 was also observed. The highest values of absorption coefficient among the products of
7-methoxyflavanone transformation were measured for 4′-hydroxy-7-methoxyflavone (4). Reduction
of the carbonyl group of 1 resulted in a decrease in absorption coefficient values in the case of 2,4-cis
and 2,4-trans 7-methoxyflavan-4-ol (2 and 3) (Table 4).
The IC50 values (antiradical activity) of the substrate and the biotransformation products were
determined spectrophotometrically on the basis of graphs: DPPH radical reduction as a function of
concentration of a tested compound [16]. IC50 means the concentration of an antioxidant (flavonoid)
that reduces the initial concentration of DPPH by 50%. The measured IC50 values are presented in
Table 5.
Table 5. The IC50 values of the 7-methoxyflavanone (1) and the biotransformation products.
Substrate
Product
IC50* (± SD) [μM]
9.50 (± 0.03)
7.66 (± 0.05)
7.75 (± 0.03)
8.20 (± 0.06)
8.42 (± 0.06)
7-Methoxyflavanone (1)
4’-Hydroxy-7-methoxyflavone (4)
4,2’-Dihydroxy-4’-methoxydihydrochalcone (5)
2,4-cis-7-Methoxyflavan-4-ol (2)
2,4-trans-7-Methoxyflavan-4-ol (3)
* Mean values of IC50 calculated as an average of at least three measurements
Among the products of the microbial transformation of 7-methoxyflavanone the highest antioxidant
activity was observed for 4'-hydroxy-7-methoxyflavone (4) (IC50 = 7.66) and 4,2'-dihydroxy-4'-
methoxydihydrochalcone (5) (IC50 = 7.75). This is due to the microbial introduction of a hydroxyl
group at 4' position in ring B. The two reduction products: 2,4-cis (2) and 2,4-trans 7-methoxyflavan-
4-ol (3) have also higher antioxidant properties than the substrate (1). Comparison of the IC50 values of
2 and 3 indicates that 2,4-cis-7-methoxyflavan-4-ol (2) is a better antioxidant (IC50 = 8.20) (Table 5),
which shows that stereochemistry of a compound may have an influence on its antioxidant activity.
Our earlier research on antioxidant activity of products of biotransformations of flavanone, its
monosubstituted derivatives (among them 7-methoxyflavanone) and naringenin allowed us to draw
more detailed conclusions concerning the relationship between flavonoid structure and antioxidant
activity [16].

Molecules 2012, 17
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3. Experimental
3.1. Analysis
The course of microbial transformation was monitored by TLC (SiO2, DC Alufolien Kieselgel 60
F254, Merck, Darmstadt, Germany). Chromatograms were developed using the following developing
systems: hexane-ethyl acetate (7:3), dichloromethane-ethyl acetate (1:1), toluene-diethyl ether (4:1).
Column chromatography (SiO2, Kieselgel 60, 230–400 mesh, 40–63 μm, Merck) was performed using
1
the same eluents. H-NMR and 13C-NMR spectra were recorded with a Bruker Avance DRX 300
spectrometer. IR spectra were determined with a Mattson IR 300 Thermo Nicolet spectrometer. Mass
spectra were obtained using high-resolution electrospray ionization (ESI+-MS) (Waters LCT Premier
XE mass spectrometer).
HPLC analyses were performed with a Waters 2690 instrument equipped with Waters 996
photodiode array detector, using ODS 2 column (4.6 × 250 mm, Waters) and a Guard-Pak Inserts
μBondapak C18 pre-column. Separation conditions were as follows: gradient elution, using 80% of
acetonitrile in 4.5% formic acid solution (eluent A) and 4.5% formic acid (eluent B); flow,
1 mL/min; detection wavelength 280 nm; program: 0–7 min, 10% A 90% B; 7–10 min, 50% A 50% B;
10–13 min, 60% A 40% B; 13–15 min, 70% A 30% B; 15–20 min 80% A 20% B; 20–30 min 90% A
10% B; 30–40 min, 100% A. Melting points were determined with a Boetius apparatus (Kofler block).
Antioxidant properties were measured with a Cintra 20 spectrometer (GBC, Melbourne, Australia).
3.2. Materials
The racemic substrate for biotransformation, 7-methoxyflavanone (1), was purchased from Aldrich
1
13
(Poznań, Poland). C16H14O3; Melting point 89–91 °C; Full description of the H-NMR and C-NMR
spectra can be found in our previous paper [20].
Microorganisms
The wild strain A. niger KB was obtained from the collection of the Department of Biotechnology
and Food Microbiology of Wrocław University of Environmental and Life Sciences (Poland). The
microorganism was maintained on potato slants (sterilized piece of potato) at 5 °C.
The wild strains A. ochraceus 456 and P. chermesinum 113 were obtained from the collection of the
Department of Chemistry of Wrocław University of Environmental and Life Sciences (Poland). The
microorganisms were maintained on agar slants at 5 °C.
3.3. Biotransformations
3.3.1. Screening Procedure
Cultivation media consisted of 3% glucose (The Industrial and Trading Enterprise “Stanlab” Co. Ltd.,
Lublin, Poland) and 1% peptobac (BTL sp. z o.o., Warszawa, Poland) in water. The microorganisms
were transferred from the slants to 500 mL Erlenmayer flasks, each containing 200 mL of the medium.
Preincubation was performed at 25 °C for 24–48 h. Then portions of 1 mL of the culture solution were

Molecules 2012, 17
14817
transferred to inoculate 500 mL flasks, each containing 200 mL of the medium. After cultivation at 25 °C
for 24 h on a rotary shaker, 10 mg of a substrate, dissolved in 0.5 mL of THF, was added to the grown
culture. Control cultivation with no substrate was also performed. After 1, 3, 6 and 9 days of
incubation under the above conditions, portions of 5 mL of the transformation mixture were withdrawn
and extracted with ethyl acetate (3 × 3 mL). The extracts were dried over MgSO4 (5 min), concentrated
in vacuo and analyzed by TLC. Quantitative analyses of the mixtures were performed by means of
HPLC. Calibration curves for quantitative analyses were prepared using isolated and purified
biotransformation products as standards.
3.3.2. Preparative Biotransformation
Portions of 1 mL of the preincubation culture solution were used to inoculate three 2000 mL flasks,
each containing 500 mL of the cultivation medium. The cultures were incubated at 25 °C for 48 h on a
rotary shaker. Then 50 mg of the substrate dissolved in 2.5 mL of THF was added to each flask
(100 mg of the substrate per 1 L of the cultivation mixture). After 9 or 10 days of incubation the
mixtures were extracted with ethyl acetate (3 × 200 mL), dried (MgSO4) and concentrated in vacuo.
The transformation products were separated by column chromatography. Pure products were identified
by means of spectral analyses (TLC, 1H-NMR, 13C-NMR, IR).
The physicochemical and spectrometric data of the products were as follows:
(±)-2,4-cis-7-Methoxyflavan-4-ol (2) [21]. C16H16O3; Melting point 116–118 °C; 76% yield; purity
23
546
1
98% (HPLC); [α] = 0, (c = 1.0, CH3OH); H-NMR (CDCl3) : 2.13 (1H, ddd, J3ax,3eq = 13.2 Hz,
J3ax,2 = 11.4 Hz, J3ax,4 = 10.2 Hz, H-3ax), 2.52 (1H, ddd, J3eq,3ax = 13.2 Hz, J3eq,4= 6.3 Hz, J3eq,2 = 2.0 Hz,
H-3eq), 3.79 (3H, s, 7-OCH3), 5.05 (1H, broad s, H-4), 5.17 (1H, dd, J2,3ax = 11.4 Hz, J2,3eq = 2.0 Hz,
H-2), 6.45 (1H, d, J8,6 = 2.5 Hz, H-8), 6.59 (1H, dd, J6,5 = 8.6 Hz, J6,8 = 2.5 Hz, H-6), 7.36 (1H, d,
J5,6 = 8.6, H-5), and 7.34–7.45 (5H, m, B-ring protons); 13C-NMR (CDCl3) : 40.2 (C-3), 55.3 (OCH3),
65.6 (C-4), 77.1 (C-2), 101.1 (C-8), 108.1 (C-6), 118.2 (C-10), 126.0 (C-2', C-6'), 127.8 (C-4'), 128.1
(C-5), 128.6 (C-3′, C-5′), 140.4 (C-1'), 155.5 (C-9), and 160.3 (C-7); IR (CH2Cl2, max, cm−1): 3225
(O-H, stretch), 1619 (C-C, stretch, aromatic), and 1590 (C-C, stretch, aromatic); HRESI-MS [M+H+]
(calculated/found) (m/z 257.0990/257.0979).
(+)-2,4-trans-7-Methoxyflavan-4-ol (3). C16H16O3; Melting point 115–117 °C; 4.9% yield; purity 97%
20
546
1
(HPLC); [α]
= +2.57, (c = 0.7, CH3OH), ee = 30%; H-NMR (CDCl3) : 2.52 (1H, ddd,
J3eq,3ax = 13.2 Hz, J3eq,4 = 6.2 Hz, J3eq,2 = 1.9 Hz, H-3eq), 2.95 (1H, ddd, J3ax,3eq = 13.1 Hz, J3ax,2 = 12.0
Hz, J3ax,4 = 3.2 Hz, H-3ax), 5.07 (1H, m, H-4), 5.17 (1H, dd, J2,3ax = 11.5 Hz, J2,3eq = 1.8 Hz, H-2), 6.45
(1H, d, J8,6 = 2.5 Hz, H-8), 6.58 (1H, dd, J6,5 = 8.4 Hz, J6,8 = 2.5 Hz, H-6), 7.42 (1H, d, J5,6 = 8.5, H-5),
13
and 7.34–7.47 (5H, m, B-ring protons); C-NMR (CDCl3) : 40.0 (C-3), 55.2 (7-OCH3), 65.6 (C-4),
77.2 (C-2), 101.2 (C-8), 108.1 (C-6), 118.0 (C-10), 126.0 (C-2', C-6'), 127.9 (C-4'), 128.2 (C-5), 128.7
(C-3', C-5'), 140.1 (C-1'), 155.0 (C-9), and 160.1 (C-7); IR (CH2Cl2, max, cm−1): 3230 (O-H, stretch),
1619 (C-C, stretch, aromatic) and 1595 (C-C, stretch, aromatic); HRESI-MS [M+H+]
(calculated/found) (m/z 257.0990/257.0977).

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4'-Hydroxy-7-methoxyflavone (4) [22]. C16H12O4; Melting point 195–197 C; 2.3% yield; purity 98%
1
(HPLC); H-NMR (CDCl3) : 3.78 (3H, s, 7-OCH3), 5.69 (1H, s, H-3), 6.51 (1H, d, J8,6 = 2.5 Hz,
H-8), 6.57 (1H, dd, J6,5 = 8.4 Hz, J6,8 = 2.5 Hz, H-6), 6.95 (2H, d, J = 8.6 Hz, H-3', H-5'), 7.42 (1H, d,
J
5,6 = 8.3 Hz, H-5), 7.81 (2H, d, J = 8.7 Hz, H-2', H-6'), and 9.88 (1H, s, 4'-OH); 13C-NMR (CDCl3) :
55.4 (7-OCH3), 101.4 (C-8), 104.0 (C-3), 108.4 (C-6), 115.9 (C-3', C-5'), 122.0 (C-1'), 126.3 (C-10),
128.1 (C-5), 128.6 (C-2', C-6'), 160.5 (C-4'), 161.2 (C-9), 162.8 (C-2 ), 168.6 (C-7), and 174.6 (C-4);
IR (KBr, max, cm−1): 3432 (O-H, stretch), 1670 (C = O, stretch), 1405 (C-C, stretch, aromatic) and 774
(C-H, bending, aromatic); HRESI-MS [M+H+] (calculated/found) (m/z 269.0965/269.0960).
4,2'-Dihydroxy-4'-methoxydihydrochalcone (5) [23]. C16H16O4; Melting point 121–123 C; 15% yield;
purity 99% (HPLC); 1H-NMR (CDCl3) : 2.98 (2H, t, J = 7.5 Hz, H-β), 3.19 (2H, t, J = 7.8 Hz, H-α),
3.83 (3H, s, 4'-OCH3), 5.38 (1H, s, 4-OH), 6.40 (1H, d, J = 2.5 Hz, H-3’), 6.42 (1H, dd, J = 9.0 Hz,
J = 2.4 Hz, H-5'), 6.77 (2H, d, J = 8.5, H-3, H-5), 7.11 (2H, d, J = 8.5, H-2, H-6), 7.63 (1H, d, J = 9.0,
H-6'), and 12.60 (1H, s, 2'-OH); 13C-NMR (CDCl3) : 29.9 (C-β), 40.1 (C-α), 55.6 (-OCH3), 101.2
(C-3'), 107.5 (C-5'), 114.0 (C-1'), 115.7 (C-3, C-5), 129.5 (C-2, C-6), 131.3 (C-6'), 133.0 (C-1), 155.1
(C-4), 166.1 (C-2'), 166.7 (C-4'), and 191.5 (C=O); IR (CH2Cl2, max, cm−1): 3576 (O-H, stretch), 1706
(C = O, stretch), 1650 (C-C, stretch, aromatic) and 1480 (C-C, stretch, aromatic); HRESI-MS [M+H+]
(calculated/found) (m/z 273.1111/273.1105).
3.4. Measurement of Antioxidant Properties of the Substrate and the Products
A methanolic solution of DPPH (1,1-diphenyl-2-picryl-hydrazyl) with an absorbance of about 1.00,
was mixed with a proper amount of a tested flavonoid 1–5. After 20 min, disappearance of absorbance
at 520 nm was measured. The initial concentration of DPPH was determined by means of calibration
curve. The IC50 value (antiradical activity) was determined graphically—DPPH radical reduction
(expressed in %) as a function of concentration of the tested compound. IC50 means concentration of
the antioxidant that reduces the initial concentration of DPPH by half.
4. Conclusions
The study on A. niger KB described in this article and in our previous papers prove that this strain is
a good catalyst for carbonyl group reduction in flavanone and its monosubstituted derivatives [19,20].
The strain P. chermesinum 113 performs mainly reactions of hydroxylation in ring B, which are often
accompanied with the ether bond cleavage in ring C, leading to dihydrochalcone structure [15].
The biotransformations of 7-methoxyflavanone did not afford any enantiomerically pure product.
Enantiomeric excess of 2,4-trans-7-methoxyflavan-4-ol obtained in the reaction catalysed by
A. ochraceus 456 did not exceed 30%.
All the products of biotransformations of 7-methoxyflavanone have higher antioxidant properties
than the substrate.

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Acknowledgments
This work was financed by the project “Biotransformations for pharmaceutical and cosmetics
industry” No.POIG.01.03.01-00-158/09, which is partly financed by the European Union within the
“European Regional Development Fund for the Innovative Economy”.
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Sample Availability: Samples of the compounds are available by Edyta Kostrzewa-Susłow from
Department of Chemistry, Wrocław University of Environmental and Life Science.
© 2012 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 license
(http://creativecommons.org/licenses/by/3.0/).