Preparation of silybin phase II metabolites: Streptomyces catalyzed glucuronidation

Article abstract of DOI:10.1016/j.molcatb.2014.02.008

Abstract Flavonolignan silybin is a major component of the silymarin complex isolated from seeds of the milk thistle (Silybum marianum) having strong antioxidant and hepatoprotective effects, and also anticancer, chemoprotective, dermatoprotective and hypocholesterolemic activities. Natural silybin (silibinin in pharmacological literature) is a mixture of two diastereomers: silybin A and silybin B. Their metabolism is strongly linked to Phase II biotransformations and respective conjugates are rapidly excreted in bile and urine. Conjugation reactions of both silybins are strictly stereoselective. Therefore, optically pure compounds must be used for metabolic studies. The aim of this study was to obtain the glucuronidated metabolites of both silybin A and B. Streptomyces sp. strain M52104 was found to be a highly effective tool for the preparation of silybin A-20-O-β-glucuronide, silybin B-20-O-β-glucuronide, silybin A-7-O-β-glucuronide and silybin B-7-O-β-glucuronide and minor amounts of silybin A and B-5-O-β- glucuronide. The glucuronides, which were thoroughly characterized by MS and NMR spectroscopy, can be used as invaluable authentic standards in metabolic studies of both silybin diastereomers.

Full text of DOI:10.1016/j.molcatb.2014.02.008

Journal of Molecular Catalysis B: Enzymatic 102 (2014) 167–173
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Preparation of silybin phase II metabolites: Streptomyces catalyzed
glucuronidation
Cedric Charriera, Robert Azerada,b,∗, Petr Marholc, Katerˇina Purchartovád,
Marek Kuzmac, Vladimír Krˇenc,∗
a Bertin-Pharma, 10 bis avenue Ampère, Parc d’Activités du Pas du Lac, 78180 Montigny-le-Bretonneux, France
b Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Université Paris Descartes, 45 rue des Sts Pères,
75006 Paris, France
c Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenˇská 1083, CZ 14220 Prague, Czech Republic
d Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Flavonolignan silybin is a major component of the silymarin complex isolated from seeds of the milk
thistle (Silybum marianum) having strong antioxidant and hepatoprotective effects, and also anticancer,
chemoprotective, dermatoprotective and hypocholesterolemic activities. Natural silybin (silibinin in
pharmacological literature) is a mixture of two diastereomers: silybin A and silybin B. Their metabolism
is strongly linked to Phase II biotransformations and respective conjugates are rapidly excreted in bile
and urine. Conjugation reactions of both silybins are strictly stereoselective. Therefore, optically pure
compounds must be used for metabolic studies. The aim of this study was to obtain the glucuronidated
metabolites of both silybin A and B. Streptomyces sp. strain M52104 was found to be a highly effective
tool for the preparation of silybin A-20-O-␤-glucuronide, silybin B-20-O-␤-glucuronide, silybin A-7-O-␤-
glucuronide and silybin B-7-O-␤-glucuronide and minor amounts of silybin A and B-5-O-␤-glucuronide.
The glucuronides, which were thoroughly characterized by MS and NMR spectroscopy, can be used as
invaluable authentic standards in metabolic studies of both silybin diastereomers.
Received 23 December 2013
Received in revised form 21 January 2014
Accepted 10 February 2014
Available online 20 February 2014
Keywords:
Silybin diastereomers
Silibinin
Silymarin
Glucuronidation
Glucuronyl transferase
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
broad interest in the detailed study of these activities. Administra-
tion of silymarin (or silybin) proved to be often beneficial for cancer
The flavonolignan silybin (CAS No. 22888-70-6; also denoted
as silibinin in pharmacological literature) and its congeners such
as 2,3-dehydrosilybin, isosilybin, silydianin and silychristin are
important natural compounds obtained from silymarin, which is
a complex extract from the seeds of Silybum marianum (L.) Gaertn.
(Asteraceae; milk thistle) [1,2]. In the last decades, silymarin and
silybin were in the focus mainly due to multiple beneficial biolog-
ical activities. Originally known for its anti-phalloidin activity [3],
and usable for the treatment of various liver disorders [4], sily-
bin and its congeners, frequently as constituents of milk thistle
extract, were later identified as antioxidants with cytoprotective
effects and also potential hypocholesterolemic drugs [5,6]. Reports
on the anticancer activities of silymarin components [7] triggered
patients either by releasing negative side effects of chemotherapy
or by potentiating anticancer treatment [2].
The molecular mechanism of silybin action is not completely
clear. Only antioxidant and antiradical mechanisms were described
in detail [8,9] showing that the C-20 OH is the most important moi-
ety for antiradical activity and the C-7 OH possesses pro-oxidant
activity, forming stable radicals.
Natural silybin is typically an approximately equimo-
lar mixture of two diastereomers (Fig. 1), silybin
(1a)—(2R,3R)-2-[(2R,3R)-2,3-dihydro-3-(4-hydroxy-3-
methoxyphenyl)-2-(hydroxymethyl)-1,4-benzodioxin-6-yl]-
2,3-dihydro-3,5,7-trihydroxy-4H-1-benzopyran-4-one—and
A
silybin
B
(1b)—(2R,3R)-2-[(2S,3S)-2,3-dihydro-3-(4-hydroxy-
3-methoxyphenyl)-2-(hydroxymethyl)-1,4-benzodioxin-6-yl]-
2,3-dihydro-3,5,7-trihydroxy-4H-1-benzopyran-4-one (Fig. 1)
(usually 1b is slightly prevalent in most preparations). Thanks
to our recently developed enzymatic methods enabling effec-
tive separation of silybin A and B in the multigram quantities
[10,11] we can perform now an array of chemical and biological
∗
Corresponding author at: Instiute of Microbiology, CZ 14220 Prague, Czech
Repblic. Tel.: +420 296442510; fax: +420 296 442 509..
E-mail addresses: robert.azerad@parisdescartes.fr (R. Azerad),
kren@biomed.cas.cz, robert.azerad@parisdescartes.fr (V. Krˇen).
http://dx.doi.org/10.1016/j.molcatb.2014.02.008
1381-1177/© 2014 Elsevier B.V. All rights reserved.

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C. Charrier et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 167–173
Fig. 1. Structures of silybin diastereomers and their glucuronides; ␤GlcA = ␤-D-glucuronyl.
experiments with pure silybin diastereomers at the preparative
scale.
of two silybin glucuronides (C-20 and C-7) was tested and proved
that 7-O-silybin-␤-glucuronide is a better antioxidant than free
silybin [14] that is in good agreement with the finding that C-7
OH in the silybin molecule has prooxidative properties [9].
Metabolic profiles of resorbed silybin indicate that it mainly
circulates in blood in the conjugated form [13,14]. Therefore, bio-
logical studies with the pure, well defined silybin conjugates are of
utmost importance.
Typical method for preparation of silybin glucuronides using
liver microsomes (with UGT activity) is quite feasible, but the
amount of product is limited by extremely high price of UDPGA.
Purification of the respective conjugates is labour and time
consuming and usually involves preparative HPLC. Chemical glu-
curonidation e.g. using Koenigs–Knorr reaction is not easy as it
involves complicated protection/deprotection methods. The use of
heavy metals (Cd, Hg, Ag) is incompatible with silybin due to its
strong complexation activity and the traces of metals are impossi-
ble to remove from the final preparations [21].
Silybin undergoes in human various biotransformation pro-
cesses and is largely metabolized by both Phase I and II enzymes.
Due to the complex nature of the silybin molecule bearing five
OH groups and four stereogenic centers, its biotransformation
processes and interactions are very complex. Stereochemistry of
silybin is of the utmost importance, as it is now clear that silybin
A (1a) and silybin B (1b) are metabolized in completely different
ways, both in terms of pharmacokinetics[12,13] and pharmacody-
namics, yielding different metabolites [14].
Beside sulfate ester formation [15], glucuronidation is one of the
main conjugation reactions of flavonoids and polyphenols [16]: it
is catalyzed by UDP-glucuronosyltransferases (UGTs), the micro-
somal enzymes encoded by a multigene family in humans.
Krˇen et al. [14] prepared the 7-O-␤-glucuronide, 20-O-␤-
glucuronide, and 5-O-␤-glucuronide of the optically pure “silybin
A” [17] by enzymatic synthesis using ovine liver microsomes and
uridine-5ꢀ-diphosphoglucuronic acid (UDPGA) as a donor, however
only at the mg scale required for metabolic profiling.
These conjugates were formed in vitro in the proportion 7-O
(62.5%), 20-O (27%), 5-O (2.5%), which reflects the approximate
regio-preference of UGTs for silybin B molecule. This [14] and later
the Han’s paper [13] are probably the only studies so far describ-
ing the full spectral and, therefore, structural evidence (1H, 13C
NMR and MS) for these important silybin conjugates/metabolites.
Krˇen et al. [14] established that silybin B 20-O-␤-glucuronide
(3b) is the major silybin B conjugate in humans, while the C-7
regioisomer is also formed, but in a lower proportion. Han [13]
demonstrated that silybin B (1b) was glucuronidated (using bovine
microsomes + UDPGA) at ca. 2–3 × higher rate than its diastere-
omer (1a), and glucuronidation of silybin B was much preferred
at the C-20, while that of silybin A was similar at both C-7 and
C-20. In the mixture, both diastereomers influence the respective
metabolism of each other [13]. Jancˇová [18] tested silybins (both 1a
and 1b) metabolite formation in vitro by incubation with a human
liver microsomal fraction, then with primary cultures of human
hepatocytes and with twelve recombinant isoenzymes of UGT.
Only three recombinant UGTs (UGT1A4, UGT2B4 and UGT2B17)
do not participate in silybin glucuronidation processes. Chen et al.
[19] investigated the glucuronidation of various flavonoids by
recombinant UGT1A3 and UGT1A9 as well. Hoh et al. [20] identi-
fied four silybin monoglucuronides and two minor diglucuronides
(HPLC–MS), however, without determining respective regioiso-
mers, in patients with colorectal carcinoma after the administration
of Silipide (Indena SpA, IT) containing silymarin (in complex with
phospholipids).
Recently, some of us [22] published a microbial transforma-
tion method enabling the glucuronidation of various polyphenolic
dietary flavonoids, such as naringenin or quercetin and some stil-
benoids, such as resveratrol, rhapontigenin or deoxyrhapontigenin.
Therefore, we have decided to apply this methodology to the
preparation of silybin glucuronides starting from the pure silybin
diastereomers.
2. Materials and methods
2.1. Melting points, optical rotation
Melting points were measured on a capillary instrument (Büchi,
CH) and are uncorrected. Optical rotations were measured in a 1 dm
cell on a Perkin Elmer 321 spectropolarimeter (GB).
2.2. NMR
In Prague (Inst. Microbiol.) NMR spectra were recorded with
a Bruker Avance III 400 MHz spectrometer (400.13 MHz for 1H,
100.55 MHz for 13C at 30 ◦C), a Bruker Avance III 600 MHz spectrom-
eter (600.23 MHz for 1H, 150.93 MHz for 13C at 30 ◦C) and a Bruker
Avance III 700 MHz spectrometer (700.13 MHz for 1H, 176.05 MHz
for 13C at 30 ◦C) in DMSO-d6 (99.9 atom% D, Sigma-Aldrich). Resid-
ual signals of the solvent were used as an internal standard (␦H
2.500 ppm, ␦C 39.60 ppm). NMR experiments: 1H NMR, 13C NMR, J-
resolved, COSY, HSQC, HSQC-TOCSY, HMBC, and 1D TOCSY were
performed using the manufacturer’s software. 1H NMR and 13C
NMR spectra were zero-filled to fourfold data points and mul-
tiplied by a window function before Fourier transformation. A
two-parameter double-exponential Lorentz–Gauss function was
applied for 1H to improve resolution, and line broadening (1 Hz)
Scarce attempts to test the biological activity of silybin glu-
curonides were limited by the paucity of the compounds and
expensive method of their preparation. Radical scavenging activity

C. Charrier et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 167–173
169
was used to obtain a better 13C signal-to-noise ratio. Chemical shifts
are given on a ␦-scale with digital resolution justifying the reported
values.
out with an Agilent DAD detector (200–400 nm) and a Quatro
Micromass spectrometer (Waters) operated in negative mode from
150 to 900 D.
In Paris (Univ. Descartes) 1H- and 13C-NMR spectra (500.13 MHz
and 125.77 MHz, respectively) were recorded on a Bruker Avance-
500 spectrometer in DMSO-d6 or acetone-d6 solvents. All NMR
spectra were measured at 20 ◦C, and proton and carbon chem-
ical shifts were referenced to the solvent signals: 2.50 ppm for
proton and 39.52 ppm for carbon in DMSO-d6, and 2.05 ppm for
proton and 29.85 ppm for carbon in (CD3)2CO. Two-dimensional
correlation spectroscopy (COSY) and total correlation spectroscopy
(TOCSY), rotating frame nuclear Overhauser effect spectroscopy
(ROESY), heteronuclear single quantum coherence (HSQC), and het-
eronuclear multiple bond correlation (HMBC) experiments were
performed using standard pulse sequences. Proton chemical shift
assignments were obtained by the analysis of COSY and TOCSY
spectra and carbon chemical shift assignments were made by the
analysis of HSQC and HMBC spectra. For reference, a complete pro-
ton and carbon peak assignment of the NMR spectra was made on
the parent compounds.
2.5. Cultivation of microorganism
The Streptomyces sp. strain M52104, isolated from soil (for the
full details of the strain used see [22–24]), was maintained on
ISP medium 2 agar (Difco) slants at 25–28 ◦C. Liquid medium for
cultures contained 2% D-(+)-glucose, 0.5% yeast extract (Difco),
0.5% soytone (Difco), 0.5% NaCl, 0.5% K2HPO4 in deionized water,
adjusted to pH 7 with HCl solution. Media were sterilized by auto-
claving at 121 ◦C for 20 min. Precultures of 50 mL were inoculated
with four drops of a bacterial cell suspension and incubated 72 h at
27 ◦C in the orbital shaker (200 rpm). Each preculture was used to
inoculate 1 L cultures, which were incubated at 27 ◦C (200 rpm) for
72 h.
2.6. Preparative syntheses and isolation of products
2.3. MALDI-TOF/TOF
Streptomyces sp. M52104 was grown at 27 ◦C with orbital shak-
ing (200 rpm) in 1L-conical flasks containing 250 mL-volume of
liquid medium during 72 h. Silybin substrates were added to the
culture (0.1 g/L final concentration) and incubations were con-
tinued for 96 h and monitored by HPLC/UV/MS. The cells were
removed by filtration on GF/A filter (Whatman) with celite fil-
ter aid, and the filtrate treated with Amberlite XAD-16. The
resin was repeatedly washed with water and then the glu-
curonides were eluted with ethanol. After evaporation in vacuo
to a small volume, the eluate residue was solubilized in a mini-
mal volume of acetonitrile and submitted to successive injections
(0.5–2 mL) on a semi-preparative Nucleodur (Macherey-Nagel)
C18 column (10 ␮m, 32 × 250 mm) using a water-acetonitrile
gradient, containing 0.1% formic acid (40 mL/min) and detec-
tion at 280 nm. Collected fractions were evaluated for purity by
HPLC/UV/MS and those fractions that contained sufficient quantity
and purity of the glucuronides of interest were pooled, evapo-
rated and lyophilized. Final purity (in the 80–100% range) was
estimated from analytical HPLC and UV absorption between 230
and 320 nm using the aglycone molecular absorption as a refer-
ence.
MALDI–MS spectra were measured on
a MALDI-TOF/TOF
ultraFLEX III mass spectrometer (Bruker-Daltonics, Bremen, DE).
Positive/negative spectra were calibrated externally using the
monoisotopic [M + H]+ or [M − H]− ions of PepMixII calibrant
(Bruker-Daltonics). ␣-Cyano-4-hydroxycinnamic acid (5 mg/mL)
in 50% CH3CN/0.1% TFA was used as a MALDI matrix. A 0.4 ␮L
of sample dissolved in MeCN was premixed with 0.5 ␮L of the
matrix solution on the target and allowed to dry at ambient
temperature. The MALDI-TOF spectra were collected in reflectron
mode. The exact masses were measured using LTQ Orbitrap XL
hybrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA,
USA) equipped with an electrospray ion source. The mobile phase
consisted of methanol/water (4:1), flow rate 30 ␮L/min and the
samples were injected using a 2-␮L loop. Spray voltage, capil-
lary voltage, tube lens voltage and capillary temperature were
4.5 kV, −40 V, −120 V and 275 ◦C, respectively. The mass spectra
were internally calibrated using deprotonated stearic acid as lock
mass.
2.4. HPLC
3. Results
Analytical chromatography was carried out on the Shimadzu
Prominence UFLC system (Kyoto, JP) consisting of a DGU-20A
mobile phase degasser, two LC-20AD solvent delivery units, a
SIL-20ACHT cooling autosampler, a CTO-10AS column oven and
SPD-M20A diode array detector. The Chromolith Performance RP-
18e monolithic column (100 × 3 mm i.d., Merck, Darmstadt, DE)
coupled with a guard column (5 × 4.6 mm) (Merck, DE) was used
in all analyses. The PDA data were acquired in the 200–450 nm
range and the 285 nm signal was extracted. Binary gradient elu-
tion: mobile phase A (CH3CN/H2O/HCOOH (10/90/0.1; v/v/v);
mobile phase B (CH3CN/H2O/HCOOH (90/10//0.1; v/v/v)); gradi-
ent, 0–1.5 min, 100% A; 1.5–15 min, 0–100% B; 16–16.5 min, 100–0%
B. Flow rate was 1 mL/min at 25 ◦C. All samples were dissolved in
water.
The separation and quantification of glucuronide metabolites
was carried out using an Agilent model 1100 liquid chromato-
graph interfaced with UV and MS detection. Separations were
performed at 40 ◦C on Nucleodur (Macherey-Nagel) C18 columns
(5 ␮m, 4 × 70 mm for preliminary separations and 3 × 250 mm for
final measurements), using a similar water-acetonitrile gradient,
containing 0.1% formic acid (0.5 mL/min). Detection was carried
As shown in Figs. 2 and 3, both silybin A (1a) and B (1b)
(100 mg each) are nearly completely converted by the Streptomyces
strain into three monoglucuronides, accounting for the presence
of 3 peaks (Figs. 2 and 3, and S25, Supplementary material) at
ca. 15.5 min (2a/2b), ca. 17.4 min (4a/4b) and 15.9 min (3a/3b)
with a similar mass of m/z 657 [M − H] and a similar fragment
at m/z 481 [M-H-176] corresponding to the aglycone fragment
(Fig. S22, Supplementary material). After separation and purifi-
cation by semipreparative HPLC, two of them, 2a/2b (34 and
26 mg, respectively), and 3a/3b (6 and 4 mg, respectively) were in
large excess compared to 4a/4b (less than 1 mg). Another minor
product, not isolated, was detected at a lower retention time (Rt
12.3 min) and exhibited mass peaks at m/z 833 (657 + 176) and
657 (Fig. S23, Supplementary material), and might correspond to
an undetermined diglucuronidated derivative. In addition, a small
peak with m/z 655 and 479 (2 AMU lower than the major glu-
curonide peaks, Fig. S24, Supplementary material) was detected
with a higher Rt (19.3 min) and a higher UV absorption (see
Figs. 2 and 3).

170
C. Charrier et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 167–173
Fig. 2. Analytical HPLC profile of silybin A biotransformation after 72 h-incubation. 1—combined single mass monitoring at m/z 657 + 481; 2—UV detection (200–400 nm).
Fig. 3. Analytical HPLC profile of silybin B derivatives after 72 h-incubation. 1—combined single mass monitoring at m/z 657 + 481; 2—UV detection (200–400 nm).
3.1. Silybin A 7-O-ˇ-glucuronide (2a)
3.4. Silybin B 20-O-ˇ-glucuronide (3b)
HPLC purity 100%, m. p. 160–164 ◦C dec, [␣]d-3.3, (c 0.155,
MeOH), 1H, 13C NMR and MS, see Supplementary data Table S1
and Fig. S22, respectively.
HPLC purity 79%, m. p. 145–148 ◦C, [␣]d-4.8 (c 0.117, MeOH), 1
H
and 13C NMR, see Supplementary data Table S2.
3.5. Silybin A 5-O-ˇ-glucuronide (4a)
HPLC purity 100%, 1H and 13C NMR, see Supplementary data
Table S1a.
3.2. Silybin B 7-O-ˇ-glucuronide (2b)
HPLC purity 94%, 1H and 13C NMR, see Supplementary data Table
S2.
3.6. Silybin B 5-O-ˇ-glucuronide (4b)
1H and 13C NMR, see Supplementary data Table S2; m. p. and
[␣]d were not measured due to compound paucity.
3.3. Silybin A 20-O-ˇ-glucuronide (3a)
4. Discussion
HPLC purity 90%, m. p. 210–215 ◦C dec., [␣]d-29.4 (c 0.015,
As the metabolism of silybin is strictly stereoselective [12] it was
instrumental to use for this conjugation reactions optically pure
MeOH), 1H and 13C NMR, see Supplementary data Table S1.

C. Charrier et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 167–173
171
compounds. We have demonstrated that microbial glucuronidation
using Streptomyces sp. strain M52104 of both 1a and 1b affords at
least three glucuronides that were earlier determined to be human
metabolites of silybin (but only for silybin B [14]). Previous method
used for preparation of minute amounts of silybin B glucuronides
([14] is rather complicated (work with active liver microsomes) and
it employs inhibitory expensive UDP-GlcA, therefore our method
presented here is not only easier and cheaper but also more flexible
as it affords, e.g. glucuronides of silybin A that were not prepared
so far.
Our method is likely to afford the conjugates of both sily-
bin diastereomers in larger amounts (hundreds of mg) enabling
thus detailed metabolic studies but also detailed studies of bio-
logical activities of respective metabolites. Furthermore, several
compounds detected in minor amount was not isolated but their UV
spectra corresponded to tentative 2,3-dehydrosilybin glucuronide
[25]. Silybin is known to undergo spontaneous oxidation at neutral
and basic conditions affording respective 2,3-dehydroderivatives
[26,27] but formation through the microbial transformation cannot
be excluded.
Silybin B 20-O-␤-glucuronide (3b) is plausibly the major sily-
bin B conjugate in humans, while the C-7 regioisomer is also
formed, but in a lower proportion [14]. Han et al. [13] demon-
strated that silybin B (1b) was glucuronidated (using bovine
microsomes + UDPGA) at ca. 2–3 times higher rate than its diastere-
omer (1a), and glucuronidation of silybin B was much preferred
at the C20-position, while that of silybin A was similar at both
C7- and C20-positions. In the mixture, both diastereomers influ-
ence the respective metabolism of each other [13]. Silybins (both
1a and 1b) were tested for the metabolite formation in vitro by
incubation with a human liver microsomal fraction, then with
primary cultures of human hepatocytes and with 12 recom-
binant isoenzymes of UGT [18]. Only three recombinant UGTs
(UGT1A4, UGT2B4 and UGT2B17) do not participate in silybin
glucuronidation processes. Chen et al. [19] investigated the glu-
curonidation of various flavonoids by recombinant UGT1A3 and
UGT1A9 as well. Four silybin monoglucuronides and two minor
diglucuronides (HPLC–MS) were identified as metabolites, how-
ever, withoutdetermining respectiveregioisomers, inpatientswith
colorectal carcinoma after the administration of Silipide (Indena
SpA, IT) containing silymarin (in complex with phospholipids)
[20].
In addition to mass spectrometry results, detailed NMR data
(Tables S1 and S2, Supplementary material) were obtained for the
main glucuronide derivatives 2a–4a, 2b–4b, allowing to assign
the glucuronidation positions from 2D-HSQC and HMBC spectra.
In 2a and 2b, clear 3J diagnostic correlations could be observed
between the anomeric H-1ꢀ and C-7 (see Figs. 4 and 5), indi-
cating a structure of silybin 7-O-␤-glucuronides, whereas in
3a and 3b three bonds 1H–13
C
3J correlations between H-1ꢀ
and C-20 were observed, indicating a structure of silybin 20-
␤-glucuronides. The ␤-configuration of the four glucuronides
was deduced from the high coupling constant H-1ꢀ/H-2ꢀ in the
glucuronide moieties (J = 7.0–7.6 Hz)—see Tables S1 and S2 (Sup-
plementary material).
The amounts of 4a and 4b were not sufficient to obtain signifi-
cant HMBC spectra but the large chemical shift difference between
H-6 and H-8 (0.38 ppm, compared with 0–0.04 ppm for the other
investigated compounds) suggests a structure of silybin 5-O-␤-
glucuronides for these compounds. As shown in Tables S1 and S2
(Supplementary material), all other ı values were correctly corre-
lated in the various products (and the initial substrate, accounting
for the change of NMR solvent).
Carefully characterized glucuronides will be used not only
as authentic standards in metabolic studies of both silybin
diastereomers but also for further synthesis of combined con-
jugates (glucuronidated and sulfated) by enzymatic methods
[27,28].
UDP-glucuronyltransferase from Streptomyces strain M52104
has not been isolated yet. DNA of respective enzyme has been not
sequenced. Generally, BLAST alignment of human UGT (GenBank
ID NM 001076) against Streptomyces genome revealed identity of
Scarce attempts to test the biological activity of silybin glu-
curonides were limited by the paucity of the compounds and
expensive method of their preparation. Radical scavenging activ-
ity of two silybin glucuronides (C-20 and C-7) was tested and
proved that 7-O-silybin-␤-glucuronide is a better antioxidant than
free silybin [14] that is in good agreement with the finding
that C-7 OH in the silybin molecule has prooxidative properties
[9].
Fig. 4. 1H −→ 13C 3J correlations observed in HMBC spectra of glucuronides 2a and 3a.

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C. Charrier et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 167–173
Fig. 5. 1H −→ 13C 3J correlations observed in HMBC spectra of glucuronides 2b and 3b.
putative proteins resulted in 20%. There are rather scarce data
on the prokaryotic UDP-glucuronyltransferases; the first one has
been isolated from Streptomyces griseochromogenes [29], then ␤-
1,4-glucuronosyltransferase GelK from the gellan gum-producing
bacillus Sphingomonas paucimobilis has been described [30]. These
limited data do not allow generalization of the structural elements
and comparison with well characterized mammal glucuronosyl-
transferases.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2014.02.008.
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ˇ
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We present here an effective production of the glucuronides
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