Enzymatic activity of cell-free extracts from Burkholderia oxyphila OX-01 bio-converts (+)-catechin and (-)-epicatechin to (+)-taxifolin

Article abstract of DOI:10.1080/09168451.2016.1220822

This study characterized the enzymatic ability of a cell-free extract from an acidophilic (+)-catechin degrader Burkholderia oxyphila (OX-01). The crude OX-01 extracts were able to transform (+)-catechin and (-)-epicatechin into (+)-taxifolin via a leucocyanidin intermediate in a two-step oxidation. Enzymatic oxidation at the C-4 position was carried out anaerobically using H₂O as an oxygen donor. The C-4 oxidation occurred only in the presence of the 2R-catechin stereoisomer, with the C-3 stereoisomer not affecting the reaction. These results suggest that the OX-01 may have evolved to target both (+)-catechin and (-)-epicatechin, which are major structural units in plants.

Full text of DOI:10.1080/09168451.2016.1220822

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Enzymatic activity of cell-free extracts from
Burkholderia oxyphila OX-01 bio-converts (+)-
catechin and (−)-epicatechin to (+)-taxifolin
Yuichiro Otsuka, Motoki Matsuda, Tomonori Sonoki, Kanna Sato-Izawa,
Barry Goodell, Jody Jelison, Ronald R. Navarro, Hitoshi Murata & Masaya
Nakamura
To cite this article: Yuichiro Otsuka, Motoki Matsuda, Tomonori Sonoki, Kanna Sato-Izawa,
Barry Goodell, Jody Jelison, Ronald R. Navarro, Hitoshi Murata & Masaya Nakamura (2016):
Enzymatic activity of cell-free extracts from Burkholderia oxyphila OX-01 bio-converts (+)-
catechin and (−)-epicatechin to (+)-taxifolin, Bioscience, Biotechnology, and Biochemistry, DOI:
10.1080/09168451.2016.1220822
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Date: 09 September 2016, At: 01:07

Bioscience, Biotechnology, and Biochemistry, 2016
Enzymatic activity of cell-free extracts from Burkholderia oxyphila OX-01
bio-converts (+)-catechin and (−)-epicatechin to (+)-taxifolin
Yuichiro Otsuka1,5,*, Motoki Matsuda2, Tomonori Sonoki3, Kanna Sato-Izawa4, Barry Goodell5,
Jody Jelison6, Ronald R. Navarro1, Hitoshi Murata7 and Masaya Nakamura1
1Department of Forest Resource Chemistry, Forestry and Forest Products Research Institute, Tsukuba, Japan;
3
2Department of Biotechnology, Toyama Prefectural University, Imizu, Japan; Faculty of Agriculture and Life
4
Science, Hirosaki University, Hirosaki, Japan; Graduate School of Life and Environmental Sciences, University of
5
Tsukuba, Tsukuba, Japan; Department of Sustainable and Biomaterials, Virginia Polytechnic and State University,
6
Blacksburg, VA, USA; Center for Agriculture, Food and the Environment, University of Massachusetts Amherst,
7
Amherst, MA, USA; Department of Mushroom Science and Forest Microbiology, Forestry and Forest Products
Research Institute, Tsukuba, Japan
Received May 31, 2016; accepted July 21, 2016
http://dx.doi.org/10.1080/09168451.2016.1220822
This study characterized the enzymatic ability of
a cell-free extract from an acidophilic (+)-catechin
degrader Burkholderia oxyphila (OX-01). The crude
OX-01 extracts were able to transform (+)-catechin
and (−)-epicatechin into (+)-taxifolin via a leuco-
cyanidin intermediate in a two-step oxidation. Enzy-
matic oxidation at the C-4 position was carried out
anaerobically using H2O as an oxygen donor. The
C-4 oxidation occurred only in the presence of the
2R-catechin stereoisomer, with the C-3 stereoisomer
not affecting the reaction. These results suggest
that the OX-01 may have evolved to target both
(+)-catechin and (−)-epicatechin, which are major
structural units in plants.
we did not clarify how (+)-catechin was being con-
verted to taxifolin by enzymatic reaction. This work
builds upon that previous research to look more closely
at the mechanisms involved in the transformation. Fur-
thermore, the stereospecificity of the enzymatic reaction
against various cathechin isomers was also evaluated.
Understanding the function of the catechin metabolic
enzymes in OX-01 will potentially contribute to our
overall understanding of the cycling of flavonoids in
forest ecosystems.
Materials and methods
Micro-organism, medium and culture conditions.
Burkholderia oxyphila OX-01 (NBRC 105797 =DSM
22550) was isolated from an acidic forest soil in Tsu-
kuba, Japan, and grown in W medium6) containing
0.2% (+)-catechin as the sole carbon source at pH 3.5
(adjusted with HCl). The culture medium was filter-
sterilized using a Millex-GV filter unit (0.22 μm, Milli-
pore Corp., Bedford, MA, USA) and OX-01 was then
grown in stationary culture, or shaken at 28 °C (50 mL
of media in a 200 mL Erlenmeyer flask). For stationary
culture, 15 g of agar (Nacalai Tesque, Inc., Kyoto,
Japan) was added to 1.0 L of the above media.
Key words: burkholderia; (+)-catechin; (+)-taxifolin;
(−)-epicatechin; enzymatic reaction
Catechin is a flavonoid and a secondary metabolite of
plants. It is a major precursor of condensed tannin, which
is the second-most abundant aromatic plant biomass fol-
lowing lignin.1) Catechin plays an important role as a
defensive material in plants,2) and is secreted from the
roots to maintain a network of micro-organisms in the
rhizosphere.3) There is significant interest in how cate-
chin, which is produced in large quantities by plants, is
decomposed and recycled by soil micro-organisms.
Preparation of crude OX-01 extract.
Cells in the
mid-log growth phase were harvested by centrifugation
at 8000 g for 20 min at 4 °C. The resulting pellet was
washed twice with 50-mM phosphate buffer (pH 7.0),
and resuspended in 2 ml of the same buffer. The sus-
pension was then homogenized using a French press
(Ohtake Seisakusho, Tokyo, Japan) at 4 °C. Unbroken
cells were removed by centrifugation (13,000 g for
10 min at 4 °C). The supernatant was stored at 4 °C
and directly used throughout the experiments.
In 2011, a bacterium identified as Burkholderia oxy-
phila OX-01 was isolated from an acidic forest soil and
was shown to grow on (+)-catechin as a sole carbon
source under acidic conditions (pH 3.5).4) Prior to that
research, only one study of bacterial (+)-catechin degra-
dation to taxifolin under acidic conditions by a
Burkolderia species had been published.5) In that paper,
we identified that Burkholderia sp. KTC-1 metabolizes
(+)-catechin via laucocyanidin and taxifolin.5) However,
*Corresponding author. Email: yotuka@ffpri.affrc.go.jp
© 2016 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
Y. Otsuka et al.
Measurement of enzymatic activity of crude OX-01
of the UV spectra, 25 μL of the crude OX-01 extract
was added to 1 mL of the different reaction mixtures
and then incubated at 30 °C.
extract. During preliminary analysis, a reaction mix-
ture containing 0.002% (+)-catechin in 50-mM phos-
phate buffer (pH 7.0) was prepared. Into 1 mL of this
solution, 25 μL of crude extract solution was added
and the UV spectra of the sample were evaluated at
28 °C. As control, the same crude extract solution,
which was boiled for 15 min to inactivate the enzymes
and then centrifuged at 13,000 g for 10 min at 4 °C,
was employed.
To elucidate the structure of the reaction product, the
reaction mixture was extracted with ethyl acetate and
purified using silica gel column chromatography with
chloroform/ethyl acetate/formic acid (10:8:1) as the
mobile phase. The purified product was analyzed by
NMR spectroscopy. NMR spectra were measured at
600 MHz with a JNM-ECX-600 instrument (JEOL,
Akishima, Japan). CD3OD was used as solvent and as
1
internal standard for H-NMR at δ3.30 ppm.
HPLC analysis of enzymatic reaction product.
One mL of the crude OX-01 extract was added to 9 mL
of the reaction mixture. At 20-min intervals, 1 mL of
the reaction solution was collected and then soon frozen
with liquid nitrogen to stop the reaction. The frozen
samples were lyophilized. Acetonitrile (0.5 mL) was
added to the lyophilized sample and the mixture was
centrifuged at 14,000 g for 10 min at 4 °C. The super-
natant was passed through a Minisart RC15 syringe fil-
ter (Sartorius AG, Göttingen, Germany) and then
subjected to HPLC analysis using a C-18 column (Inert-
sil ODS-3, 5 μm column, 4.6 × 250 mm, GL Sciences
Inc., Tokyo, Japan) with detection by UV/Vis detector
(UV970, JASCO Corp., Japan) set at 280 nm. Exactly
20 μL of sample was injected and eluted for 15 min at a
flow rate of 1.0 mL/min of solvent A (10 mM H3PO4
sol.) with a linear gradient from 10 to 50% of solvent B
(acetonitrile).
Results
Analysis of the (+)-catechin metabolic pathway by a
crude OX-01 extract reaction
Preliminary evaluation of the enzymatic activity of
crude OX-01 extracts from Burkholderia oxyphila
OX-01 for (+)-catechin degradation has revealed posi-
tive results. As shown in Fig. 1(A), the UV spectra of
the treated sample showed striking shifts in the absor-
bance values relative to the initial sample to indicate
the chemical conversion of (+)-cathechin. The treated
sample also displayed an increase in absorbance at
325 nm suggesting the formation of a new compound.
In this connection, the isolation of the enzyme by
ammonium sulfate precipitation as well as gel perme-
ation chromatography was attempted. Unfortunately,
despite our significant efforts, product isolation was
found to be extremely difficult as even a slight indica-
tion of enzymatic activity was not detected. Even
attempts to incorporate possible cofactors in the reac-
tion were unsuccessful. In any case, with the major
goal of describing the degradation pathway of (+)-cate-
chin in natural environments, the observed activity
prompted us to further evaluate various parameters in
relation to its activity against this compound.
To elucidate the pathway for (+)-catechin metabolism
in B. oxyphila OX-01, the intermediate and by-products
of the action of the crude OX-01 extract at different
reaction times were analyzed by HPLC. Figure 2 shows
the time course of enzymatic reaction with (+)-catechin.
The peak representing (+)-catechin rapidly decreased
and was completely undetected after 60 min. This was
accompanied by the appearance of a new major peak
representing taxifolin, which increased with the decline
of (+)-catechin peak. In addition, another peak that was
identified in our previous study,5) which represents leu-
cocyanidin, was also found to increase initially and
then subsequently decreased during the reaction pro-
cess. These results suggest that (+)-catechin was being
Incorporation of 18O from H218O.
The reaction
was initiated by the addition of 10 μL of the crude
OX-01 extract into the reaction mixture containing 100
μL of 100 mM phosphate buffer (pH 7.0), 2 μL of
0.2% (+)-catechin and 100 μL of 18O2-labeled water
(99 atom%18O; Taiyo Nippon Sanso Co., Tokyo,
Japan) or non-labeled water. The mixtures were incu-
bated for 15 h at 30 °C after which the product was
extracted with ethyl acetate and then evaporated. The
residue was dissolved in 50 μL of pyridine and treated
with 50 μL of BSA + TMCS + TMSI 3:2:3 (Supelco)
for 30 min at 65 °C to prepare trimethylsilyl (TMS)
derivatives. Two μL of this solution was subjected to
GC–MS analysis (1200 Quadrupole Mass Spectrome-
ters, Varian Inc., California, USA) to assess the incor-
poration of the labeled water. A factor-four VF-5MS
column (30 m × 0.25 mm ID, DF 0.25 mm) was used
as the stationary phase. The temperature of the mobile
phase was increased from 180 to 300 °C at a rate of
10 °C/min
converted into (+)-taxifolin via
a
leucocyanidin
intermediate by the crude OX-01 extract obtained from
OX-01.
Analysis of substrate-specific enzymatic activity of
crude OX-01 extract.
Four stereoisomers (+)-cate-
chin, (−)-catechin, (+)-epicatechin, and (−)-epicatechin
reacted with the crude OX-01 extract. All substrates
were purchased from Nagara Science (Gifu, Japan).
The enzyme reaction and measurement of UV spectra
were conducted as described in a previous study by
Matsuda et al.5). Briefly, the reaction mixtures contain-
ing 0.002% of catechin stereoisomer in 50-mM phos-
phate buffer (pH 7.0) were prepared. For measurement
Effect of dissolved oxygen and incorporation of 18O
from H218O
More detailed evaluation of the mechanism for enzy-
matic reaction was conducted to determine the oxygen
donor for (+)-taxifolin formation. Initially, the possible
contribution of dissolved oxygen in generating carbonyl
groups was considered. For this purpose, the crude

Enzymatic anaerobic oxidation of catechins
3
OH
OH
OH
OH
(B)
(A)
1.6
HO
HO
S
R
R
O
O
1.6
S
OH
OH
OH
OH
0.8
0
0.8
0
230
280
330
OH
230
280
Wavelength (nm)
330
Wavelength (nm)
OH
OH
(D)
OH
(C)
HO
HO
S
S
R
R
O
O
1.6
1.6
OH
OH
OH
OH
0.8
0.8
0
0
230
280
330
230
280
330
Wavelength (nm)
Wavelength (nm)
Fig. 1. Substrate specific properties of the C-4 oxidative activity of crude enzyme. The crude enzyme of OX-01 reacted with (+)-catechin (A),
(−)-catechin (B), (+)-epicatechin (C), and (−)-epicatechin (D).
Note: Squares show the UV spectra of the substrate (■) and the reaction product (□).
H218O during enzymatic reaction. The TMS-modified
products were analyzed by GC–MS. The major product,
trimethylsilyl-taxifolin (TMS-taxifolin), was detected at
18.3 min albeit at different mass values (m/z). The reac-
tion product with non-labeled H2O was detected as the
m/z 664.5 (Fig. 3(A)) molecular ion. On the other hand,
the reaction product with H218O was detected as two m/
z 664.3 and 666.4 molecular ions (Fig. 3(B)). One of
the fragment ions (fragment I) derived from the A-ring
including the C-4 carbonyl group was detected as (m/z
296.1 and 298.1, data are not shown). Another fragment
ion (fragment II) derived from the B-ring did not incor-
porate the H218O.
Stereoselectivity of catechin 4-carbonylation
To understand the stereoselectivity of carbonyl for-
mation activity, crude OX-01 extract reactions were
carried out using four different catechin stereoisomers
as substrates. After 3 h, both the UV spectra of the
reaction mixtures containing (−)-epicatechin and
(+)-catechin displayed the characteristic peak at about
325 nm (Fig. 1(A) and (D)). The UV spectra of the
reaction mixtures containing (−)-catechin and (+)-epi-
catechin showed that these enantiomers were not modi-
fied (Fig. 1 (B) and (C)).
Unexpectedly, both products from (+)-catechin and
(−)-epicatechin showed same 1H NMR spectra as
follows, δ: 6.95 (1H, d, J = 2.0 Hz, H-2′), 6.84 (1H,
dd, J = 2.0, 8.0 Hz, H-6′), 6.79 (1H, d, J = 8.0 Hz,
H-5′), 5.92 (1H, d, J = 2.0 Hz, H-6), 5.87 (1H, d,
J = 2.0 Hz, H-8), 4.90 (1H, d, J = 11.3 Hz, H-2), 4.49
(1H, d, J = 11.3 Hz, H-3). These spectra were
Fig. 2. HPLC analysis of reaction products from (+)-catechin by a
crude enzyme mixture from OX-01.
Note: The time on the left of each chromatogram represents the
reaction time.
OX-01 extract reaction was initiated after bubbling
nitrogen through the mixture to eliminate traces of oxy-
gen in the system. Despite this condition, UV analysis
of the reacted sample showed an increase in absorbance
at 325 nm indicating that carbonyl formation was not
affected by the dissolved oxygen in the system (data
not shown).
Next, we determined whether water is directly being
utilized for the oxidation of (+)-catechin. This was eval-
uated by isotope probing using labeled and non-labeled

4
Y. Otsuka et al.
(A)
Spectrum 1A
BP 368.1 (3.344e+6=100%) taxifolin002.xms
17.205 min. Scan: 1372 100.0:800.0> Ion: NA RIC: 1.419e+7 (BC)
368.1
3.344e+6
100%
75%
50%
25%
0%
369.2
2.072e+6
(A)
(B)
370.3
664.4
807305
760270
649.3
547884
650.4
371.2
327866
296.1
259293
179.0
200375
635.3
169871
561.2
354.2
239.2
101016
52996
46043
40081
100
200
300
400
500
600
700
800
m/z
(B)
Spectrum 1A
BP 368.2 (4.656e+6=100%) taxifolin001.xms
17.205 min. Scan: 1386 100.0:800.0> Ion: NA RIC: 1.990e+7 (BC)
368.2
4.656e+6
100%
75%
50%
25%
0%
369.2
2.327e+6
370.2
1.227e+6
666.5
557424
372.2
179.0
203897
652.4
298.1
258294
200468
561.4
59168
443.1
154576
54951
100
200
300
400
500
600
700
800
m/z
Fig. 3. Mass spectra of taxifolin, the products of the crude OX-01 extract reaction from (+)-catechin. Reaction products (taxifolin) of (+)-catechin
generated by the crude OX-01 extract in non-labeled water (A), and in 18O labeled water (B).
annotated as (+)-taxifolin. Therefore, these results sug-
gest that the epimerizing reaction occurs during the
conversion of (−)-epicatechin to (+)-taxifolin.
was isolated from an acidic forest soil.4) In this work, a
crude OX-01 extract was demonstrated to convert
(+)-catechin into (+)-taxifolin via a leucocyanidin inter-
mediate. The enzymatic conversion of (+)-catechin was
the same as the enzymatic reaction of Burkholderia sp.
KTC-1 that was previously reported by Matsuda
et al.5). The primary enzyme reactions for (+)-catechin
of OX-01 and KTC-1 were apparently the same and
thus a common bacterial function may be extant in at
Discussion
In a previous study, a novel (+)-catechin-degrading
bacterium, designated as Burkholderia oxyphila OX-01,

Enzymatic anaerobic oxidation of catechins
5
OH
OH
OH
OH
H
O
O
HO
O
H
O
OH
OH
OH
(+)-catechin (2R, 3S)
H2O
(-)-epicatechin (2R, 3R)
H2O
OH
OH
OH
H2
H2
OH
HO
O
HO
O
OH
OH
OH OH
OH OH
2,3-cis-leucocyanidin
2,3-trans-leucocyanidin
OH
OH
H2
H2
HO
O
O
OH
OH
(-)-epitaxifolin
OH
OH
minor
OH
OH
HO
O
HO
O
O
OH
OH
major
OH OH
OH
Compound A
Non-enzymic keto-enol tautomeric reaction
(+)-taxifolin (2R, 3S)
further degradation
Fig. 4. Proposed metabolic pathway for (+)-catechin and (−)-epicatechin to (+)-taxifolin by crude enzyme of OX-01.
least these two species permitting the conversion of
(+)-catechin under acidic conditions. Furthermore, Jef-
fery et al.7,8) reported the A-ring fission of taxifolin by
(+)-catechin-degrading bacteria isolated from rat feces,
but they did not elucidate the metabolic pathway from
(+)-catechin to taxifolin. It must be emphasized that the
enzymatic function of OX-01 was clearly different from
the (+)-catechin dioxygenase reaction that generates
protocatechuic acid and phloroglucinol carboxylic acid
To determine the oxygen donor for (+)-catechin con-
version, experiments were performed under several
conditions. No relationship was found between dis-
solved oxygen and the extent of (+)-catechin oxidation
activity when incubations were conducted using the
crude OX-01 extract. Moreover, GC–MS analysis of
the reaction mixture containing H218O, showed that
18O was incorporated into (+)-taxifolin as the carbonyl
oxygen indicating that H2O is the oxygen donor in
(+)-catechin 4-hydroxylation. These results suggest that
the oxidation of (+)-catechin by OX-01 is anaerobic
reaction using water as the oxygen donor.
from (+)-catechin via
a single-step oxidation, as
reported by Mahadevan et al.9–15). It is also distinct
from very recent findings on the biotransformation of
four cathechin stereoisomers via ring cleavage at the C-
ring oxygen by isoflavone-metabolizing bacteria in
anaerobic condition, which was found accelerated by
the presence of hydrogen.16,17)
Enzymatic carbonyl formation into C-4 occurred not
only with (+)-catechin but also with (−)-epicatechin
(Fig. 4). Enzymatic activity on (−)-catechin or (+)-
epicatechin was not observed. The (+)-catechin is a 2R,

6
Y. Otsuka et al.
3S structure and (−)-epicatechin is a 2R, 3R structure
indicating that C-4 carbonylation by OX-01 is specific to
the 2R-stereoisomer.
observation of potential relevance to the progression of
Alzheimer’s disease. These and others reports suggest
that (+)-taxifolin will continue to be a compound of
significant pharmaceutical interest. If we can utilize the
enzyme function of OX-01 effectively, (+)-taxifolin
could be produced from (+)-catechin and/or (−)-epicat-
echin, both of which are abundant plant flavonoids and
easily recovered by simple extraction. Therefore, we
believe this study will contribute not only to a better
understanding of microbial degradation of catechin in
forest ecosystems, but will also provide insights into
processes which may enable the economically favorable
production of (+)-taxifolin for commercial use.
The 1H NMR spectrum of reaction products trans-
formed from (−)-epicatechin was consistent with the
spectrum of (+)-taxifolin and not (−)-epitaxifolin. This
result indicates that the isomerization occurs at C-3
position. (Figure 4) shows the proposed conversion
pathway of (+)-catechin and/or (−)-epicatechin into
(+)-taxifolin in the presence of a cellular extract of
OX-01. This crude OX-01 extract not only converted
(+)-catechin to (+)-taxifolin but also was capable
of converting (−)-epicatechin to (+)-taxifolin. The
(−)-epicatechin was converted to (−)-epitaxifolin via a
2,3-cis-leucocyanidin
intermediate
because
the
Author contributions
stereoisomers at the C-3 position were stable when the
2,3-cis-leucocyanidin was formed.18) The (−)-epitaxi-
folin converged with (+)-taxifolin as a more stable
structure via compound A through a non-enzymatic
keto–enol tautomeric reaction.
Yuichiro Otsuka, Motoki Matsuda, Tomonori Sonoki,
Kanna Sato-Izawa, Barry Goodell, Jody Jelison, Hitoshi
Murata, and Masaya Nakamura designed this study.
Yuichiro Otsuka, Motoki Matsuda, and Hitoshi Murata
performed the experiment. Yuichiro Otsuka, Motoki
Matsuda, Barry Goodell, Jody Jellison, and Ronald R.
Navarro wrote this manuscript. All the authors
reviewed and approved the manuscript.
In aerobic bacteria, (−)-epicatechin metabolism has
not been reported. Almost all of the flavanols in free
form in the leaves, such as tea leaves, have been found
to be in the (+)-catechin (2R, 3S) structure. However,
flavanol units observed in bark as condensed tannins
were of the (+)-catechin and (−)-epicatechin (2R, 3R)
structure.19) In our work, (−)-catechin and (+)-epicate-
chin were unable to react with the crude OX-01 extract,
but it should be noted that these forms are rare in nat-
ure. There are however, some bacterium that can
degrade (−)-catechin. Wang et al.20) reported that a bac-
terium isolated from the rhizosphere of the (−)-cate-
chin-producing plant, Rhododendron formosanum,
could degrade (−)-catechin and was capable of using
(−)-catechin as a sole carbon source. The catechin
oxidative enzymes of OX-01, which lack the ability to
react with the (−)-catechin, may have a different evolu-
tionary origin and ecological function from that of the
(−)-catechin-degrading bacteria described by Wang.
Jeremy et al.21) reported that procyanidin oligomers,
constructed of catechin units, automatically decom-
posed to catechin monomers under acidic conditions. In
acidic forest soil containing leaf litter and various other
plant constituents, part of the condensed tannin also
would be automatically decomposed to catechins. This
suggests that the evolutionary targets for the OX-01
catechin oxidative enzymes were not only (+)-catechin
in leaves and nutshells as a free form, but also the
more recalcitrant (−)-epicatechins present in bark as a
condensed tannin polymer. A more detailed analysis of
the catechin degradation abilities in OX-01 will con-
tribute to the understanding of how flavonoids and con-
densed tannins, produced in great quantities by plants
are recycled by soil micro-organisms under acidic con-
ditions.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Grant-in-Aid for Young Scientists
(B) of KAKENHI [grant number 19880035]; [grant number
21780169].
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The (+)-taxifolin is not only of ecological interest
but is also of proven and potential medical interest.
This compound is not mutagenic and less toxic than
the similar flavonoid quercetin.22) Numerous reports on
(+)-taxifolin demonstrate its many medicinal applica-
23)
tions including anti-cancer
and antioxidant activ-
ity.24) In addition, Sato et al.25) recently reported that
(+)-taxifolin could inhibit amyloid β aggregations, an

Enzymatic anaerobic oxidation of catechins
7
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