Biotransformation of Chrysin to Baicalein: Selective C6-Hydroxylation of 5,7-Dihydroxyflavone Using Whole Yeast Cells Stably Expressing Human CYP1A1 Enzyme

Article abstract of DOI:10.1021/acs.jafc.7b02690

Naturally occurring polyphenolic compounds are of medicinal importance because of their unique antioxidant, anticancer, and chemopreventive properties. Baicalein, a naturally occurring polyhydroxy flavonoid possessing a diverse range of pharmacological activities, has been used in traditional medicines for treatment of various ailments. Apart from its isolation from natural sources, its synthesis has been reported via multistep chemical approaches. Here, we report a preparative-scale biotransformation, using whole yeast cells stably expressing human cytochrome P450 1A1 (CYP1A1) enzyme that allows regioselective C6-hydroxylation of 5,7-dihydroxyflavone (chrysin) to form 5,6,7-trihydroxyflavone (baicalein). Molecular modeling reveals why chrysin undergoes such specific hydroxylation mediated by CYP1A1. More than 92% reaction completion was obtained using a shake-flask based process that mimics fed-batch fermentation. Such highly efficient selective hydroxylation, using recombinant yeast cells, has not been reported earlier. Similar CYP-expressing yeast cell based systems are likely to have wider applications in the syntheses of medicinally important polyphenolic compounds.

Full text of DOI:10.1021/acs.jafc.7b02690

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Article
Biotransformation of chrysin to baicalein: Selective C6-
hydroxylation of 5,7-dihydroxyflavone using whole
yeast cells stably expressing human CYP1A1 enzyme
Ibidapo S Williams, Shifali Chib, Vijay Nuthakki, Linda Gatchie, Prashant Joshi, Niteen
Narkhede, Ram A. Vishwakarma, Sandip B. Bharate, Saurabh Saran, and Bhabatosh Chaudhuri
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02690 • Publication Date (Web): 07 Aug 2017
Downloaded from http://pubs.acs.org on August 14, 2017
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Biotransformation of chrysin to baicalein: Selective C6-
hydroxylation of 5,7-dihydroxyflavone using whole yeast
cells stably expressing human CYP1A1 enzyme
†
,‡,#
§,#
#
†,‡
⊥
⊥
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Ibidapo S. Williams,
Shifali Chib, Vijay Nuthakki, Linda Gatchie, Prashant Joshi, Niteen A.
ǁ
,φ,*
⊥
§,
,†,‡
⊥
Narkhede, Ram A. Vishwakarma, Sandip B. Bharate,
Saurabh Saran, * Bhabatosh Chaudhuri*
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# Contributed equally to this work as a first author.
†
Leicester School of Pharmacy, De Montfort University, Leicester, LE1 9BH, UK
CYP Design Limited, Innovation Centre, 49 Oxford Street, Leicester, LE1 5XY, UK
‡
§
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Fermentation Technology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road,
Jammu-180001, India.
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⊥Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-
180001, India
ǁ
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Instrumentation Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180001,
India
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φAcademy of Scientific & Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine,
Canal Road, Jammu-180001, India
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ABSTRACT:
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Naturally occurring polyphenolic compounds are of medicinal importance because of their unique
antioxidant, anticancer and chemopreventive properties. Baicalein, a naturally occurring polyhydroxy
flavonoid possessing a diverse range of pharmacological activities, has been used in traditional
medicines for treatment of various ailments. Apart from its isolation from natural sources, its synthesis
has been reported via multiꢀstep chemical approaches. Here we report a preparativeꢀscale
biotransformation, using whole yeast cells stably expressing human cytochrome P450 1A1 (CYP1A1)
enzyme, that allows regioselective C6ꢀhydroxylation of 5,7ꢀdihydroxyflavone (chrysin) to form 5,6,7ꢀ
trihydroxyflavone (baicalein). Molecular modelling reveals why chrysin undergoes such specific
hydroxylation mediated by CYP1A1. More than 92% reaction completion was obtained using a shakeꢀ
flask based process that mimics fedꢀbatch fermentation. Such highly efficient selective hydroxylation,
using recombinant yeast cells, has not been reported earlier. Similar CYPꢀexpressing yeast cellꢀbased
systems are likely to have wider applications in the syntheses of medicinally important polyphenolic
compounds.
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Keywords: Biotransformation, chrysin, baicalein, CYP1A1, yeast cells, hydroxylation
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INTRODUCTION
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Baicalein (5,6,7ꢀtrihydroxyflavone) (1) was originally isolated from the roots of Scutellaria baicalensis
(Chinese skullcap) which is used for treatment of chronic hepatitis, inflammatory diseases, tumors, and
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diarrhea in China, Korea, Taiwan, and Japan. Its isolation has also been reported from another plant,
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Oroxylum indicum (Indian trumpet flower). Baicalein is known for its anticancer properties in various
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cancer types
cancer, and breast cancer.
protects against oxidative stress, more specifically in cardiac cells, and in cisplatinꢀinduced acute
including pancreatic, gastric and colorectal cancers, multiple myeloma, head and neck
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Baicalein is also considered as an antiꢀinflammatory
that generally
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kidney injury. Because of its wideꢀranging medicinal applications, an industrial scale protocol for its
production would be considered as important.
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Baicalein has primarily been produced via five approaches: (a) singleꢀstep hydrolysis of the natural
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product baicalin, a glycoside, (b) 4ꢀstep total synthesis from a cinnamic acid derivative, (c) 4ꢀstep
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total synthesis from 3,4,5ꢀtrimethoxyphenol,
(d) 3ꢀstep total synthesis from 2,4,5,6ꢀ
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tetrahydroxyacetophenone, and (e) 6ꢀstep synthesis from chrysin involving methylation, bromination,
acylation, nucleophilic replacement of Br with OMe, followed by deꢀacylation and demethylation as key
steps.
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Selective transformation, using efficient biocatalysts, of a low value phytochemical to high value
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phytochemical is an exciting area of bioorganic chemistry. Sordon and coworkers have reported
biotransformations of natural flavonoids naringenin, hesperetin, chrysin, apigenin, luteolin, quercetin,
epicatechin, and biochanin A using the natural yeast, Rhodotorula glutinis. This approach produced
norwogonin (5,7,8ꢀtrihydroxyflavone) from chrysin (5,7ꢀdihydroxyflavone). Here we report a
preparative scale process for biotransformation of chrysin to baicalein, via selective C6ꢀhydroxylation
using recombinant human cytochrome P450ꢀ1A1 (CYP1A1) enzyme expressed within baker’s yeast
(Saccharomyces cerevisiae) cells. This is the first singleꢀstep protocol for a highꢀyield conversion of
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chrysin to baicalein and, therefore, may serve as a simple and cheap strategy for production of baicalein
in an industrial scale.
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MATERIALS AND METHODS
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General. All chemicals were obtained from SigmaꢀAldrich and were used as received. H NMR spectra
were recorded on BruckerꢀAvance DPX FTꢀNMR 400 MHz instrument. Chemical data for protons are
reported in parts per million (ppm) downfield from tetramethylsilane and are referenced to the residual
proton in the NMR solvent (CD OD, 3.31 ppm). ESIꢀMS were recorded on Waters QTOF mass
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spectrometer.
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HPLC analysis was performed on Shimadzu LCꢀ6AD system connected with C18 column (4.6 x 25 mm,
5 µ). Mobile phase consisted of 0.1% formic acid (A) and methanol (B) using isocratic elution (30: 70 –
A: B). Flow rate was 1 mL/min and detection wavelength was 270 nm.
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LCꢀMS analysis was performed on Waters Acquity UPLC system. The column used was C18, 1.7 µ
with dimensions of 100 x 2.1 mm (column temp. 30 °C). Binary gradient system was used. Mobile
phase A consisted of 5% acetonitrile in water (with 0.1% formic acid). Mobile phase B consisted of
acetonitrile with 0.1% formic acid. Gradient details are: Time in min (% B concentration): 0.01 (10),
0.25 (10), 9.00 (100), 10.00 (100), 11.00 (10), 12.00 (10). PDA range: 220 nm to 400 nm; flow rate: 0.3
mL/min.
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Biotransformation experiment. Yeast strains, each of which contain two copies of human CYP1A1 or
CYP1A2 genes, downstream of the ADH2 promoter, integrated into chromosomal loci of the genome of
the yeast strain W303ꢀ1a (ATCC 208352), were used for biotransformation (Section S2 of supporting
information). Expressed CYP1A1 and CYP1A2 proteins were confirmed by Western blotting
(supporting informationꢀS4). The strains, from frozen stocks, were revived in 250 mL Erlenmeyer
baffled flasks containing 50 mL YPD (Yeast, Peptone, Dextrose) medium with composition (g/L):
peptone 20; yeast extract 10; glucose 15.0, pH 6.0. The flasks were shaken at 200 rpm, at 28 °C. Three
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consecutive YPD preꢀcultures were grown for high biomass production, before addition of the substrate
to cells grown in SD (Synthetic Defined) medium. Typically, loopful of CYPꢀcontaining freshly grown
yeast cells was inoculated in a 500 mL Erlenmeyer baffled flask separately containing 100 mL YPD
medium (preꢀculture ꢀ1) at 28 °C for 24 h. The cells were harvested after 24 h and inoculated into a new
500 mL baffled flask containing 100 mL YPD medium (preꢀculture ꢀ2) at 30 °C for 18 h. The process
was repeated three times for the cells to reach an OD , of ~90.
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The harvested cells, ~3.0 mL (OD , ~90), were inoculated in 50 mL of minimal SD medium contained
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in a 1 L baffled flask. Composition of SD medium (g/L): dextrose 1.0; dipotassium phosphate 7.0;
monopotassium phosphate 2.0; sodium citrate 0.50; magnesium sulphate 0.10; ammonium sulphate 1.0,
pH 7.0 ± 0.2 at 28 °C. Initially, reaction was carried out with 0.2 mg/mL of chrysin. Later, the chrysin at
different concentrations viz. 0.2, 1, 2, 5 and 10 mg/mL were incubated in 50 mL of SD medium (DMSO
was used for initial dissolution of the compound, and keeping the DMSO concentration < 0.5% in final
reaction medium) for 72 h at 28 °C, 200 rpm. After every 24 h, the medium was replenished with 1.5%
w/v of glucose. For optimization of incubation time, the SD cell culture media were harvested after 24,
48, 72, 96, 120, 144 and 160 h, and were then analysed on TLC and HPLC/ LCꢀMS to monitor the
yields of biotransformation at each time point.
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Isolation and characterization of baicalein. The reaction media was extracted with ethyl acetate (3
times). The combined ethyl acetate layer was concentrated on vacuoꢀrotavapor to obtain crude extracts
that contained the biotransformation product. The crude residue was loaded on a reverse phase (C18)
silica gel column packed in water. The crude extract was loaded on the column by making a slurry with
C18 silica gel mesh 200ꢀ400 (SigmaꢀAldrich, product no. 377635). The column was then eluted with
increasing concentrations of methanol in water. The desired product was collected at 50% methanol in
water. Evaporation of the solvent gave a yellow solid which was characterized as baicalein (1). Yellow
powder; m.p. 262ꢀ265 °C (Lit. 264ꢀ265 °C); TLC: R = 0.5 (3% methanol in DCM) and 0.8 (40%
f
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EtOAc: hexane with 0.1% acetic acid); H NMR (CD OD, 400 MHz, δ ppm): 7.91 (dd, J = 4.0, 8.0 Hz,
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2H), 7.48 (m, 3H), 6.66 (s, 1H), 6.55 (s, 1H); ESIꢀMS: m/z 271.10 [M+H] . The spectral data was
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identical to that reported in the literature and TLC matched with reference sample obtained from
SigmaꢀAldrich (CAS number 491ꢀ67ꢀ8).
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Molecular modeling: The docking of chrysin with CYP1A1 (PDB ID: 4I8V) was performed using
GLIDE module of Schrodinger molecular modeling software, using the protocols as described in our
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earlier publications.
The docking protocol was validated by docking known ligand αꢀ
naphthoflavone (ANF). The interaction pattern of docked ANF and ligand from coꢀcrystallized protein
(4I8V) are shown in the supporting information (Section S8).
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RESULTS AND DISCUSSION
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CYP enzymes are known for their exceptional ability to carry out hydroxylation, epoxidation or
demethylation reactions in a regioselective fashion, both in plants and humans. It is essential for human
CYP450 enzymes to be integrated on the endoplasmic reticular (ER) membranes to manifest its native
activity. Baker’s yeast cells possess ER membranes which are not present in prokaryotic E. coli cells.
Eukaryotic baker’s yeast cells were chosen because they can grow as rapidly as bacterial cells and they
are as amenable as bacteria for scaleꢀup in fermentors. Using Baker's yeast cells, we have developed an
efficient technology for stable expression of human CYP enzymes within these cells. Using this
platform, recombinant baker’s yeast cells can continue to express CYPs in shake flasks, over a week or
more, with increasing activities, under conditions that mimic fedꢀbatch growth (Supporting Information;
S1). Microsomal CYP enzymes, isolated from these same recombinant yeast cells, have successfully
been used by us earlier as drug discovery tools for screening synthetic compounds and natural product
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repositories to identify possible cancer chemopreventive agents.
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Selection of CYPs for biotransformation of chrysin. In this work, use of recombinant CYPꢀ
expressing whole yeast cells has been explored for their ability to be used as biocatalysts for
biotransformation reactions. For this purpose, we chose the flavonoid chrysin as a substrate. As the
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cytochrome P450 family 1 enzymes CYP1A1 and CYP1A2 are reported to oxidize dietary flavonoids,
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herein we chose two yeastꢀexpressed enzymes of the CYP1 subꢀfamily (i.e. CYP1A1 and CYP1A2)
as biocatalysts.
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Two recombinant yeast strains, each containing two chromosomally integrated copies of CYP1A1 and
CYP1A2 genes under the control of the ethanolꢀinducible alcohol dehydrogenase 2 (ADH2) promoter,
were created to enable stable and reproducible biotransformation reactions (plasmid maps shown in
Supporting Information, S2). The ADH2 promoter is repressed in the presence of glucose. The aim was
that recombinant yeast cells, containing stably integrated CYP expression cassettes, would be grown
initially in complete YPD medium (containing 2% glucose) to obtain large optical density, measured at a
wavelength of 600 nm (OD ) of ~90, in shake flasks. This would occur without any plasmid loss and
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would be achieved over 3 days through repetitive replenishment of glucose, every 24 h, in the nonꢀ
selective highly nutritious YPD medium. Cells grown in YPD (Figure 1a) would then be reꢀsuspended
in minimal selective SD medium (pH 7.0), supplemented with 1.5% of glucose, for the
biotransformation reaction. It was thought that rich full YPD medium may not be appropriate for
biotransformation since there is a possibility of substrate binding to its ingredients.
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For initial optimization, reactions were performed in SD with 10 mg of chrysin in baffled flasks shaking
at 200 rpm for 72 h, at 28 °C (Figure 1b). After every 24 h, the medium was replenished with fresh
glucose to a final concentration of 1.5%. Glucose was exhausted after 12 h of growth of yeast cells when
it was converted to ethanol. Hence, the ethanolꢀinducible ADH2 promoter was fully induced every 12 h
before more glucose was added to the medium for further rounds of expression.
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Figure 1.
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The reaction media at each time point was extracted with ethyl acetate. The extracts were analyzed by
TLC followed by HPLC/ LCꢀMS. The formation of new product on TLC with lower R value than the
f
substrate gave us an indication of a hydroxylation reaction (Supporting Information, S3). LCꢀMS
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analysis of the reaction mixture confirmed the product as monoꢀhydroxy chrysin with mass of m/z 270.
The same product was formed using both the enzymes, CYP1A1 and CYP1A2, expressed within yeast
cells. However, conversion of substrate to product was better with CYP1A1. Therefore, for subsequent
scaleꢀup experiments, only CYP1A1ꢀexpressing yeast cells were used for biotransformation.
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Scale up and optimization of biotransformation reaction. Scaleꢀup was performed as above; cells
were at first cultivated in nonꢀselective YPD media for 72 h, with fresh glucose (2%) being added every
24 h. Cells were reꢀsuspended in selective SD minimal medium. Chrysin at different concentrations viz.
0.2, 1, 2, 5 and 10 mg/mL were dissolved in DMSO and further incubated in 50 mL of SD medium
(keeping the DMSO concentration < 0.5%) for 72 h at 28 °C, 200 rpm. The chrysinꢀcontaining cell
culture media were replenished with glucose every 24 h to a final concentration of 1.5%. The reaction
using chrysin at concentration of 2 mg/mL (100 mg of chrysin) was observed to be the most efficient.
With 5 and 10 mg/mL concentrations, incomplete biotransformation occurred, probably because the
small number of cells, used for growth in these specific experiments, was inhibited by the substrate.
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Based on these results, it was decided to optimize the time period for biotransformation. For this, we
chose 2 mg/mL concentration of substrate with reaction time points of 24, 48, 72, 96, 120, 144 and 160
h. HPLC analyses of the reaction mixture after 24, 48, 72, 96, 120, 144 and 160 h incubations indicated
that, at 144 h, there was nearly complete conversion (>92%) of chrysin to the product. The HPLC
chromatograms of reaction mixtures at representative four time intervals are shown in Figure 2. In
HPLC analysis, the concentration of sample injected at each time interval was kept constant. The
increase in the AUC of baicalein with increase in the time, from 24 h to 144 h, is indicative of the fact
that the highest amount of baicalein is formed at the 144 h time point.
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Figure 2
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The relative percentages (AUC) of chrysin and baicalein at different time intervals are depicted in Figure
3. As shown in Figure 3, the percentage conversion of chrysin to baicalein increased with time.
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Similarly, Figure 3b shows increased peak height (mAU) of baicalein with increase in reaction time.
Figures 3a and 3b, in combination, indicate that 144 h is the optimal reaction time for this
transformation. Further details of HPLC analysis are provided in supporting information, section S5.
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Figure 3.
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Isolation and characterization of baicalein. Initial isolation attempts using normal phase silica gel
column chromatography showed significant loss in the product yield. Therefore, we attempted reverse
phase C18 silica gel column chromatography where waterꢀmethanol was used as the mobile phase. The
product was isolated at 50% methanol in water and was characterized by spectral analysis and by
comparison of its TLC with reference sample.
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The H NMR of chrysin contains a typical bunch of three singlets at chemical shift values in the range of
6 to 7 ppm. These three singlets at δ 6.24, 6.49 and 6.74 ppm correspond to the protons present at C6,
C8 and C3 positions. The C6ꢀproton appears with an upꢀfield shift in comparison to two other aromatic
protons at C3 and Cꢀ6, because of the shielding effect from two adjacent C5 and C7 hydroxyls. It is
obvious that the hydroxylation reaction is possible, either on A or C ring. Since there was no change in
the chemical shift value pattern of C ring (δ 7.91, dd, 2H and δ 7.48, m, 3H), it would indicate that the
Cꢀring is intact and no hydroxylation had taken place on this ring. The hydroxylation on A ring has two
possibilities, either C6ꢀ or C8 hydroxylation. Hydroxylation at C6ꢀposition will form baicalein (5,6,7ꢀ
trihydroxyflavone) whereas hydroxylation at C8ꢀposition will form norwogonin (5,7,8ꢀ
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trihydroxyflavone) as a product. It was interesting to see that, in the H NMR of the obtained product,
the upꢀfield singlet (δ 6.24 ppm) disappeared. This singlet peak in chrysin corresponds to the proton
present at C6ꢀposition. This gave us a clear indication that hydroxylation occurred at the C6ꢀposition,
which means that the product is 5,6,7ꢀtrihydroxyflavone. This is the naturally occurring flavone,
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commonly named as ‘baicalein’. Furthermore, on comparison of the H NMR of the obtained product
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with norwogonin, the possibility of norwogonin as the product was ruled out. In literature, the
biotransformation of chrysin using natural yeast, Rhodotorula glutinis yielded Cꢀ8 hydroxylated product
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norwogonin,
however in the present study, Cꢀ6 hydroxylated product (baicalein) was formed, which
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may be possibly because of the regioꢀspecificity of the CYP1A1 enzyme.
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Mass analysis of the isolated product showed m/z peak at 271 in ES+ve mode, which matched with the
predicted product. The final confirmation of the assigned product was done by coꢀTLC and HPLC
analysis with the reference standard of baicalein (CAS number: 491ꢀ67ꢀ8)’ purchased from Sigmaꢀ
Aldrich. TLC images as well as HPLC analysis clearly matched the reference standard (TLC images are
shown in Supporting Information ꢀS3).
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The LCꢀMS analysis was also performed for the extract as well as isolated baicalein (Figure 4).
Figure 4.
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Docking of chrysin with CYP1A1. In order to decipher the rationale for regioselective hydroxylation,
chrysin was docked with the substrate binding site of CYP1A1 enzyme (PDB ID: 4I8V). The interaction
pattern of chrysin with CYP1A1 is depicted in Figure 5. It is interesting to note that the Aꢀring of
chrysin orients towards the heme. Furthermore, the Cꢀ6 carbon of Aꢀring is present in closeꢀproximity
with heme protein, suggesting that the reactive hemeꢀoxo intermediate should possibly form at this
position. Other key interactions which help in stabilizing this orientation of chrysin includes: (a)
hydrophobic πꢀπ interactions of Pheꢀ224 of Iꢀhelix with B and C rings; (b) πꢀπ interactions of Pheꢀ123
with A ring; and (c) polar Hꢀbonding of Cꢀ7 hydroxyl group with Serꢀ122. This observed orientation,
excludes the possibility of hydroxylation at Cꢀ8 and at aromatic CH of B and C rings.
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Figure 5.
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Our efforts using yeast whole cells have resulted in the development of a reproducible preparativeꢀscale
biotransformation process for the conversion of chrysin to baicalein (5,6,7ꢀtrihydroxyflavone).
According to the literature, the medicinal effects of baicalein are more profound than that of chrysin.
Furthermore, commercially available baicalein is at least 60ꢀtimes more expensive than chrysin. Thus,
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this protocol described here can be utilized for production of a high value phytochemical from a low
value one, using a simple, lowꢀcost, oneꢀstep biotransformation reaction.
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In conclusion, we have demonstrated the ability of whole yeast cells, that overexpress the human
CYP1A1 enzyme, to catalyse biotransformation of >92% of the natural flavonoid chrysin to baicalein.
Optimal aeration, neutral pH and maintenance of glucose concentration, throughout the reaction, played
very important roles in the biotransformation reaction. The example demonstrated in this paper,
provides an opportunity for further exploring the utility of stable recombinant CYP enzymeꢀexpressing
yeast cells for industrial production of medicinally important polyphenolic compounds.
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ASSOCIATED CONTENT
Supporting Information. Additional experimental details. This material is available free of charge via
the Internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
*Tel:
sandipbharate@gmail.com (S.B.B.)
*Tel: +91ꢀ191ꢀ2585006. Fax: +91ꢀ191ꢀ2586333. Eꢀmail: ssaran@iiim.ac.in (SS)
+91ꢀ191ꢀ2585006.
Fax:
+91ꢀ191ꢀ2586333.
Eꢀmail:
sbharate@iiim.ac.in,
*Tel: 44(0)116 250 7280; Fax: +44(0) 116 257 7287; Eꢀmail: bchaudhuri@dmu.ac.uk (B.C.)
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250
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253
254
255
ORCID
Ram A. Vishwakarma: 0000ꢀ0002ꢀ0752ꢀ6238
Sandip B. Bharate: 0000ꢀ0001ꢀ6081ꢀ5787
Author Contributions
The enzyme expression in yeast cells was executed by I. S. Williams, L. Gatchie and B. Chaudhuri. Preꢀ
culturing of yeast cells and their growth in SD medium followed by setꢀup of biotransformation
experiment was performed by S. Chib and S. Saran. Product isolation and characterization was done by
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V. Nuthakki, R.A. Vishwakarma and S. B. Bharate. LSꢀMS analysis was done by N.A. Narkhede and
molecular modeling by P. Joshi and S.B. Bharate.
Funding
th
This work was supported by CSIR 12 five year plan project BSCꢀ0205 (S.B.B.) and Higher Education
Innovation Fund (HEIF) (B.C.).
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Notes
The authors declare no competing financial interest.
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64
ABBREVIATIONS
265
266
267
268
269
ADH2, alcohol dehydrogenase 2; ANF, alphaꢀnaphthoflavone; CYP1A1, cytochrome P450 group
enzyme 1A1; CYP1A2, cytochrome P450 group enzyme 1A2; DMSO, dimethyl sulfoxide; ER,
endoplasmic reticulum; HPLC, high performance liquid chromatography; LCꢀMS, liquid
chromatography mass spectrometry; OD, optical density; PDB, protein data bank; SD, Synthetic
Defined; TLC, thinꢀlayer chromatography; YPD, Yeast, Peptone, Dextrose;
2
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71
2
ACKNOWLEDGMENT
th
2
2
72
73
SBB and BC thank CSIR 12 FYP project BSCꢀ0205 and Higher Education Innovation Fund (HEIF) for
financial support.
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REFERENCES
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276
277
1.
Papafragkakis, C.; Ona, M. A.; Reddy, M.; Anand, S. Acute Hepatitis after Ingestion of a
Preparation of Chinese Skullcap and Black Catechu for Joint Pain. Case Reports Hepatol. 2016,
2016, Article ID: 4356749.
12
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Journal of Agricultural and Food Chemistry
Roy, M. K.; Nakahara, K.; Na, T. V.; Trakoontivakorn, G.; Takenaka, M.; Isobe, S.; Tsushida, T.
Baicalein, a flavonoid extracted from a methanolic extract of Oroxylum indicum inhibits
proliferation of a cancer cell line in vitro via induction of apoptosis. Pharmazie 2007, 62, 149ꢀ
153.
278
279
280
281
2.
2
2
2
82
83
84
3.
4.
Zhou, R. T.; He, M.; Yu, Z.; Liang, Y.; Nie, Y.; Tai, S.; Teng, C. B. Baicalein inhibits pancreatic
cancer cell proliferation and invasion via suppression of NEDD9 expression and its downstream
Akt and ERK signaling pathways. Oncotarget 2017, DOI: 10.18632/oncotarget.16912.
285
286
287
288
Wang, Y. F.; Xu, Y. L.; Tang, Z. H.; Li, T.; Zhang, L. L.; Chen, X.; Lu, J. H.; Leung, C. H.; Ma,
D. L.; Qiang, W. A.; Wang, Y. T.; Lu, J. J. Baicalein Induces Beclin 1ꢀ and Extracellular Signalꢀ
Regulated KinaseꢀDependent Autophagy in Ovarian Cancer Cells. Am J Chin Med 2017, 45,
123ꢀ136.
289
290
291
5.
PalkoꢀLabuz, A.; SrodaꢀPomianek, K.; Uryga, A.; KostrzewaꢀSuslow, E.; Michalak, K.
Anticancer activity of baicalein and luteolin studied in colorectal adenocarcinoma LoVo cells
and in drugꢀresistant LoVo/Dx cells. Biomed Pharmacother 2017, 88, 232ꢀ241.
2
2
92
93
6.
7.
Chai, Y.; Xu, J.; Yan, B. The antiꢀmetastatic effect of baicalein on colorectal cancer. Oncol Rep
2017, 37, 2317ꢀ2323.
294
295
296
Bie, B.; Sun, J.; Li, J.; Guo, Y.; Jiang, W.; Huang, C.; Yang, J.; Li, Z. Baicalein, a Natural Antiꢀ
Cancer Compound, Alters MicroRNA Expression Profiles in Belꢀ7402 Human Hepatocellular
Carcinoma Cells. Cell Physiol Biochem 2017, 41, 1519ꢀ1531.
2
97
98
8.
9.
Rui, X.; Yan, X. I.; Zhang, K. Baicalein inhibits the migration and invasion of colorectal cancer
2
cells via suppression of the AKT signaling pathway. Oncol Lett 2016, 11, 685ꢀ688.
299
300
301
Nguyen, L. T.; Song, Y. W.; Cho, S. K. Baicalein Inhibits Epithelial to Mesenchymal Transition
via Downregulation of Cyr61 and LOXLꢀ2 in MDAꢀMB231 Breast Cancer Cells. Mol Cells
2016, 39, 909ꢀ914.
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 25
3
02
03
10.
11.
Mu, J.; Liu, T.; Jiang, L.; Wu, X.; Cao, Y.; Li, M.; Dong, Q.; Liu, Y.; Xu, H. The Traditional
3
Chinese Medicine Baicalein Potently Inhibits Gastric Cancer Cells. J Cancer 2016, 7, 453ꢀ461.
304
305
306
Ma, X.; Yan, W.; Dai, Z.; Gao, X.; Ma, Y.; Xu, Q.; Jiang, J.; Zhang, S. Baicalein suppresses
metastasis of breast cancer cells by inhibiting EMT via downregulation of SATB1 and Wnt/betaꢀ
catenin pathway. Drug Des Devel Ther 2016, 10, 1419ꢀ1441.
3
3
3
07
08
09
12.
13.
14.
Choi, E. O.; Park, C.; Hwang, H. J.; Hong, S. H.; Kim, G. Y.; Cho, E. J.; Kim, W. J.; Choi, Y.
H. Baicalein induces apoptosis via ROSꢀdependent activation of caspases in human bladder
cancer 5637 cells. Int J Oncol 2016, 49, 1009ꢀ1018.
310
311
312
Cathcart, M. C.; Useckaite, Z.; Drakeford, C.; Semik, V.; Lysaght, J.; Gately, K.; O'Byrne, K. J.;
Pidgeon, G. P. Antiꢀcancer effects of baicalein in nonꢀsmall cell lung cancer inꢀvitro and inꢀvivo.
BMC Cancer 2016, 16, 707.
313
314
315
Guo, Z.; Hu, X.; Xing, Z.; Xing, R.; Lv, R.; Cheng, X.; Su, J.; Zhou, Z.; Xu, Z.; Nilsson, S.; Liu,
Z. Baicalein inhibits prostate cancer cell growth and metastasis via the caveolinꢀ1/AKT/mTOR
pathway. Mol Cell Biochem 2015, 406, 111ꢀ119.
3
16
17
15.
16.
Zheng, Y. H.; Yin, L. H.; Grahn, T. H.; Ye, A. F.; Zhao, Y. R.; Zhang, Q. Y. Anticancer effects
3
of baicalein on hepatocellular carcinoma cells. Phytother Res 2014, 28, 1342ꢀ1348.
3
3
3
18
19
20
Li, H. L.; Zhang, S.; Wang, Y.; Liang, R. R.; Li, J.; An, P.; Wang, Z. M.; Yang, J.; Li, Z. F.
Baicalein induces apoptosis via a mitochondrialꢀdependent caspase activation pathway in T24
bladder cancer cells. Mol Med Rep 2013, 7, 266ꢀ270.
321
322
323
324
17.
Kim, D. H.; Hossain, M. A.; Kang, Y. J.; Jang, J. Y.; Lee, Y. J.; Im, E.; Yoon, J. H.; Kim, H. S.;
Chung, H. Y.; Kim, N. D. Baicalein, an active component of Scutellaria baicalensis Georgi,
induces apoptosis in human colon cancer cells and prevents AOM/DSSꢀinduced colon cancer in
mice. Int J Oncol 2013, 43, 1652ꢀ1658.
14
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Page 15 of 25
Journal of Agricultural and Food Chemistry
Kim, S. J.; Kim, H. J.; Kim, H. R.; Lee, S. H.; Cho, S. D.; Choi, C. S.; Nam, J. S.; Jung, J. Y.
Antitumor actions of baicalein and wogonin in HTꢀ29 human colorectal cancer cells. Mol Med
Rep 2012, 6, 1443ꢀ1449.
325
326
327
18.
328
329
330
19.
Chen, C. H.; Huang, T. S.; Wong, C. H.; Hong, C. L.; Tsai, Y. H.; Liang, C. C.; Lu, F. J.; Chang,
W. H. Synergistic antiꢀcancer effect of baicalein and silymarin on human hepatoma HepG2
Cells. Food Chem Toxicol 2009, 47, 638ꢀ644.
3
31
32
20.
21.
Motoo, Y.; Sawabu, N. Antitumor effects of saikosaponins, baicalin and baicalein on human
3
hepatoma cell lines. Cancer Lett 1994, 86, 91ꢀ95.
333
334
335
Bonham, M.; Posakony, J.; Coleman, I.; Montgomery, B.; Simon, J.; Nelson, P. S.
Characterization of chemical constituents in Scutellaria baicalensis with antiandrogenic and
growthꢀinhibitory activities toward prostate carcinoma. Clin. Cancer Res. 2005, 11, 3905ꢀ3914.
336
337
338
339
22.
Ma, Z.; Otsuyama, K.; Liu, S.; Abroun, S.; Ishikawa, H.; Tsuyama, N.; Obata, M.; Li, F. J.;
Zheng, X.; Maki, Y.; Miyamoto, K.; Kawano, M. M. Baicalein, a component of Scutellaria radix
from HuangꢀLianꢀJieꢀDuꢀTang (HLJDT), leads to suppression of proliferation and induction of
apoptosis in human myeloma cells. Blood 2005, 105, 3312ꢀ3318.
3
40
41
23.
24.
25.
Liu, H.; Dong, Y.; Gao, Y.; Du, Z.; Wang, Y.; Cheng, P.; Chen, A.; Huang, H. The Fascinating
3
Effects of Baicalein on Cancer: A Review. Int J Mol Sci 2016, 17, pii: E1681.
3
42
43
Gao, Y.; Snyder, S. A.; Smith, J. N.; Chen, Y. C. Anticancer properties of baicalein: a review.
3
Med Chem Res 2016, 25, 1515ꢀ1523.
344
345
346
Li, J.; Ma, J.; Wang, K. S.; Mi, C.; Wang, Z.; Piao, L. X.; Xu, G. H.; Li, X.; Lee, J. J.; Jin, X.
Baicalein inhibits TNFꢀalphaꢀinduced NFꢀkappaB activation and expression of NFꢀkappaBꢀ
regulated target gene products. Oncol Rep 2016, 36, 2771ꢀ2776.
15
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 25
3
3
3
47
48
49
26.
27.
28.
29.
30.
31.
Patwardhan, R. S.; Sharma, D.; Thoh, M.; Checker, R.; Sandur, S. K. Baicalein exhibits antiꢀ
inflammatory effects via inhibition of NFꢀkappaB transactivation. Biochem Pharmacol 2016,
108, 75ꢀ89.
350
351
352
Tsai, K. L.; Hung, C. H.; Chan, S. H.; Shih, J. Y.; Cheng, Y. H.; Tsai, Y. J.; Lin, H. C.; Chu, P.
M. Baicalein protects against oxLDLꢀcaused oxidative stress and inflammation by modulation of
AMPKꢀalpha. Oncotarget 2016, 7, 72458ꢀ72468.
353
354
355
Zhao, F.; Fu, L.; Yang, W.; Dong, Y.; Yang, J.; Sun, S.; Hou, Y. Cardioprotective effects of
baicalein on heart failure via modulation of Ca(2+) handling proteins in vivo and in vitro. Life
Sci 2016, 145, 213ꢀ223.
356
357
358
Sahu, B. D.; Mahesh Kumar, J.; Sistla, R. Baicalein, a Bioflavonoid, Prevents CisplatinꢀInduced
Acute Kidney Injury by UpꢀRegulating Antioxidant Defenses and DownꢀRegulating the MAPKs
and NFꢀkappaB Pathways. PLoS One 2015, 10, e0134139.
359
360
361
Zhang, W.; Yi, D.; Gao, K.; Liu, M.; Yang, J.; Liao, X.; Yang, B. Hydrolysis of scutellarin and
related glycosides to scutellarein and the corresponding aglycones. J. Chem. Res. 2014, 38, 396ꢀ
398.
362
363
364
365
366
Kim, D. H.; Yun, C. H.; Kim, M. H.; Kumar, C. N.; Yun, B. H.; Shin, J.ꢀS.; An, H. J.; Lee, Y.
H.; Yun, Y. D.; Rim, H.ꢀK.; Yoo, M.ꢀS.; Lee, K.ꢀT.; Lee, Y. S. 4'ꢀBromoꢀ5,6,7ꢀ
trimethoxyflavone represses lipopolysaccharideꢀinduced iNOS and COXꢀ2 expressions by
suppressing the NFꢀkB signaling pathway in RAW 264.7 macrophages. Bioorg. Med. Chem.
Lett. 2012, 22, 700–705.
3
67
68
32.
Chen, D. Z.; Yang, J.; Yang, B.; Wu, Y. S.; Wu, T. Total synthesis of baicalein. J Asian Nat
Prod Res 2010, 12, 124ꢀ128.
3
16
ACS Paragon Plus Environment

Page 17 of 25
Journal of Agricultural and Food Chemistry
3
3
3
69
70
71
33.
34.
35.
36.
Vyas, B.; Singh, M.; Kaur, M.; Silakari, O.; Bahia, M. S.; Singh, B. Pharmacophore and
dockingꢀbased hierarchical virtual screening for the designing of aldose reductase inhibitors:
synthesis and biological evaluation. Med Chem Res 2016, 25, 609–626.
372
373
374
Righi, G.; Antonioletti, R.; Silvestri, I. P.; D’Antona, N.; Lambusta, D.; Bovicelli, P. Convergent
synthesis of mosloflavone, negletein and baicalein from crysin. Tetrahedron 2010, 66, 1294–
1298.
3
3
3
75
76
77
Sordon, S.; Madej, A.; Popłoński, J.; Bartmańska, A.; Tronina, T.; Brzezowska, E.; Juszczyk, P.;
Huszcza†, E. Regioselective orthoꢀHydroxylations of Flavonoids by Yeast. J. Agric. Food Chem.
2016, 64, 5525−5530.
3
3
3
3
78
79
80
81
Joshi, P.; McCann, G. J. P.; Sonawane, V. R.; Vishwakarma, R. A.; Chaudhuri, B.; Bharate, S.
B. Identification of Potent and Selective CYP1A1 Inhibitors via Combined Ligand and
StructureꢀBased Virtual Screening and Their in Vitro Validation in Sacchrosomes and Live
Human Cells. J. Chem. Inform. Model. 2017, 57, 1309–1320.
382
383
384
385
386
37.
Horley, N. J.; Beresford, K. J. M.; Chawla, T.; McCann, G. J. P.; Ruparelia, K. C.; Gatchie, L.;
Sonawane, V. R.; Williams, I. S.; Tan, H. L.; Joshi, P.; Bharate, S. S.; Kumar, V.; Bharate, S. B.;
Chaudhuri, B. Discovery and characterization of novel CYP1B1 inhibitors based on heterocyclic
chalcones: Overcoming cisplatin resistance in CYP1B1ꢀoverexpressing lines. Eur. J. Med.
Chem. 2017, 129, 159ꢀ174.
3
3
3
87
88
89
38.
39.
Mohd Siddique, M. U.; McCann, G. J. P.; Sonawane, V. R.; Horley, N.; Gatchie, L.; Joshi, P.;
Bharate, S. B.; Jayaprakash, V.; Sinha, B. N.; Chaudhuri, B. Quinazoline derivatives as selective
CYP1B1 inhibitors. Eur. J. Med. Chem. 2017, 130, 320ꢀ327.
390
391
392
Mohd Siddique, M. U.; McCann, G.; Sonawane, V.; Horley, N.; Williams, I. S.; Joshi, P.;
Bharate, S. B.; Jayaprakash, V.; Sinha, B. N.; Chaudhuri, B. Biphenyl urea derivatives as
selective CYP1B1 inhibitors. Org. Biomol. Chem. 2016, 14, 8931ꢀ8936.
17
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Page 18 of 25
3
93
94
40.
41.
Okate, Y.; Walle, T. Oxidation of the flavonoids galangin and kaempferide by human liver
3
microsomes and CYP1A1, CYP1A2, and CYP2C9. Drug Metab. Disp. 2002, 30, 103ꢀ105.
395
396
397
398
Hamada, M.; Satsu, H.; Ashida, H.; SugitaꢀKonishi, Y.; Shimizu, M. Metabolites of galangin by
2,3,7,8ꢀtetrachlorodibenzoꢀpꢀdioxinꢀinducible cytochrome P450 1A1 in human intestinal
epithelial Cacoꢀ2 cells and their antagonistic activity toward aryl hydrocarbon receptor. J. Agric.
Food Chem. 2010, 58, 8111ꢀ8118.
399
400
401
42.
Ciolino, H. P.; Daschner, P. J.; Yeh, G. C. Dietary flavonols quercetin and kaempferol are
ligands of the aryl hydrocarbon receptor that affect CYP1A1 transcription differentially. Biochem
J. 1999, 340, 715ꢀ722.
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4
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FIGURE LEGENDS
Figure 1. (a) CYP1A1ꢀexpressing cells grown in YPD Medium in testꢀtubes; (b) Biotransformation
reaction using CYP1A1 in SD medium in shakeꢀflasks; (c) scheme showing conversion of chrysin to
1
baicalein, and their key H NMR chemical shift values
Figure 2. HPLC analysis of biotransformation reaction at different time intervals. (a) HPLC
chromatogram of chrysin reference standard; (b) HPLC chromatogram of baicalein reference standard;
(c) HPLC chromatogram of biotransformation reaction after 24 hrs of incubation time; (d) HPLC
chromatogram of biotransformation reaction after 48 hrs of incubation time; (e) HPLC chromatogram of
biotransformation reaction after 96 hrs of incubation time; (f) HPLC chromatogram of biotransformation
reaction after 144 hrs of incubation time. The concentration of sample injected is kept constant at each
time interval; therefore an increase in AUC (and peak height) with the increase in incubation time
indicates progress of the reaction.
Figure 3. (a) Relative percentage (±SD) of chrysin and baicalein at different time intervals during a
typical biotransformation reaction (the percentages are based on the AUC of the peaks in HPLC analysis
at 270 nm). (b) Baicalein (t = 5.99 min) peak height (±SD) at different time intervals during
R
biotransformation reaction.
Figure 4. LCꢀMS analysis of reaction mixture at 144 h. (a) LC chromatogram of reaction mixture after
1
44 h of incubation. (B) LC chromatogram of isolated product; (c) Mass spectra of peak at t 4.69 min.
R
Figure 5. Molecular docking of chrysin with CYP1A1 (PDB: 4I8V), showing the predicted site of
hydroxylation as C6.
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(
a)
(b)
4
'
4'
3'
δ 6.49
8
δ 6.56
(
c)HO
1
O
1
O
C
2'
8
C
2'
HO
HO
3'
1
'
1'
2
2
3
CYP1A1
7
7
A
B
A
B
3 δ 6.67
6
δ 6.74
δ 6.24
4
5
4
5
OH
O
OH
O
Chrysin
Baicalein
Figure 1.
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3
2
1
000
(d)
Baicalein
(a)
2
1
1
00
50
00
Chrysin
000
000
0
Chrysin
48 hrs
5
0
0
0
.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
0
300
.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
3
000 (b)
(
e)
Baicalein
Baicalein
2
1
000
000
0
2
1
00
00
0
96 hrs
Chrysin
0
.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
0
.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
44 hrs
17.5
1
1
50 (c)
500
400
Baicalein
(f)
Baicalein
2
4 hrs
00
3
2
1
00
00
00
0
1
Chrysin
5
0
0
Chrysin
0
.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
0
.0
2.5
5.0
7.5
10.0
12.5
15.0
Figure 2.
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1
1
20
00
80
60
40
20
0
Chrysin
Baicalein
2
4
48
72
96
120
144
168
Biotransformation reaction time (hrs)
(a)
6
5
4
3
2
1
00
00
00
00
00
00
0
2
4
48
72
96 120 144 168
Biotransformation reaction time (hrs)
(b)
Figure 3.
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(a)
Baicalein
Chrysin
(b)
Baicalein
(c)
[
M-H]-
Baicalein (mol wt = 270)
[
M+H]+
Figure 4.
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Figure 5.
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TOC graphic
4
'
4'
δ 6.49
8
δ 6.56
1
O
1
O
C
8
C
HO
3'
HO
HO
3'
1
'
CYP1A1-
1'
2
2
3
7
2'
7
2'
A
B
A
B
expressing
yeast cells
3 δ 6.67
6
δ 6.74
δ 6.24
4
5
4
5
OH
O
OH
O
Chrysin
Baicalein
CYP1A1
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