A rapid and simple chromatographic separation of diastereomers of silibinin and their oxidation to produce 2,3-dehydrosilybin enantiomers in an optically pure form

Article abstract of DOI:10.1055/s-0032-1328703

Silybin A and B were separated from commercial silibinin using the preparative HPLC method. The described method is rapid and effective in obtaining gram-scale amounts of two diastereoisomers with minimal effort. In our approach, silibinin was dissolved in THF (solubility greater than 100 mg/mL), an alternative solvent to H₂O or MeOH in which silibinin has a very low solubility (ca 0.05-1.5 mg/mL), and then separated into its two components using preparative RP-HPLC. By starting with purified diastereoisomers, it was possible to obtain the two enantiomers of 2,3-dehydrosilybin in good yields and optically pure using an efficient oxidation procedure. All of the purified products were fully characterised using NMR (¹H, ¹³C), CD, [α]_D, and ESI MS analyses. The purities of the products, which were evaluated using analytical HPLC, were greater than 98 % in all cases. Georg Thieme Verlag KG Stuttgart New York.

Full text of DOI:10.1055/s-0032-1328703

Letters 1077
duus marianus L., Asteraceae; milk thistle). It is an important
and natural hepatoprotective drug (Flavobion™, Legalon™) that
is widely used in the treatment of cirrhosis, chronic hepatitis,
and liver diseases associated with excessive alcohol consumption
and exposure to environmental toxins [1]. Previously, it was de-
termined that silibinin exists in two diastereoisomeric forms,
A Rapid and Simple Chromatographic
Separation of Diastereomers of
Silibinin and Their Oxidation to
Produce 2,3-Dehydrosilybin
"
namely silybin A and B [2] (1A and 1B in l Fig. 1), in a ratio of ap-
proximately 1:1 with differing stereochemistries at positions C-
Enantiomers in an Optically Pure Form 7′′ and C-8′′ in the lignan moiety [3,4]. Silibinin is a natural com-
pound with multiple biological activities operating at various cell
Giovanni Di Fabio, Valeria Romanucci, Cinzia Di Marino,
Lorenzo De Napoli, Armando Zarrelli
Department of Chemical Sciences, University of Napoli ‘Federico II’,
Napoli, Italy
levels. In the last decade, silibinin has received attention due to
its alternative beneficial activities, which mostly include anti-
cancer and chemopreventive actions, as well as cardioprotective,
neuroprotective, and antiviral activities [1,5–11]. Milk thistle ex-
"
tract also contains 2,3-dehydrosilybin (2A and 2B in l Fig. 1),
Abstract
which has antioxidant activity superior to that of silibinin and a
positive effect on some skin diseases (e.g., psoriasis, atopic ecze-
ma) [12–15]. Because of the low content of 2,3-dehydrosilybin in
its natural state, this compound was practically neglected in
studies on the biological activity of silibinin and silymarin; there-
fore, simple procedures were developed to obtain 2,3-dehydro-
silybin [16,17] and its analogues [18,19].
A major problem hampering studies of optically pure silibinin is
the extremely complicated separation of its diastereoisomers.
This problem has been approached in past years by several re-
search groups through the use of HPLC methods. Recently, a pre-
parative HPLC method for the separation of silybin A, silybin B,
and other silymarin congeners in larger quantities was described
[20,21]. A multifaceted approach has been proposed by Křen et
al. in which the chromatographic separation is performed on the
mixture of the two diastereoisomers suitably derivatised by
chemical or enzymatic methods [22–24]. Generally, these meth-
ods require special expertise in both organic synthesis and enzy-
matic kinetic resolutions. By careful analysis of the studies cur-
rently reported, it is the opinion of the authors that a rapid and
less labourious method for the separation of silibinin is still re-
quired.
!
Silybin A and B were separated from commercial silibinin using
the preparative HPLC method. The described method is rapid
and effective in obtaining gram-scale amounts of two diastereo-
isomers with minimal effort. In our approach, silibinin was dis-
solved in THF (solubility greater than 100 mg/mL), an alternative
solvent to H2O or MeOH in which silibinin has a very low solubil-
ity (ca 0.05–1.5 mg/mL), and then separated into its two compo-
nents using preparative RP-HPLC. By starting with purified dia-
stereoisomers, it was possible to obtain the two enantiomers of
2,3-dehydrosilybin in good yields and optically pure using an ef-
ficient oxidation procedure. All of the purified products were
fully characterised using NMR (1H, 13C), CD, [α]D, and ESI MS anal-
yses. The purities of the products, which were evaluated using
analytical HPLC, were greater than 98% in all cases.
Key words
prep‑RP‑HPLC · flavonolignans · Silybum marianum · Astera-
ceae · silibinin · silybin A · silybin B · 2,3‑dehydrosilybin
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/plantamedica
Here, we report a rapid and preparative separation (gram-scale
amounts) of the diastereoisomers of silibinin using preparative
HPLC (l Fig. 1) and their oxidation to produce enantiomers of
"
Silibinin is the major flavonolignan found in silymarin, which is
isolated from the fruits of Silybum marianum (L.) Gaertn. (Car-
2,3-dehydrosilybin in a very pure form using our optimised pro-
cedure [25]. Previous investigations have shown that the solubil-
Fig. 1 Separation of two diastereoisomers of sili-
binin (1A and 1B) and their oxidation to 2,3-dehy-
drosilybin A (2A) and 2,3-dehydrosilybin B (2B).
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1078 Letters
Fig. 2 Prep-HPLC profile of the silibinin. Silybin A
(1A, tR = 67.5 min) and silybin B (1B, tR = 74.5 min).
Conditions: Phenomenex Gemini C18-110A pre-
parative column (10-µm particle size, 250 mm ×
21.2 mm i.d.), eluted with H2O/MeOH/MeCN
(60:35:5, v/v/v) containing 0.1% of TFA; flow rate
12 mL/min, monitored at 288 nm.
Fig. 3 HPLC profile of sequential injections.
ity of silibinin in THF at 25°C (greater than 140–150 mg/mL) is
solution and then injected. The resolution of silybin A and silybin
B was high enough to obtain pooled fractions containing practi-
cally pure single isomers. Using groups of 10 injections at a time,
fractions of silybin A (tR = 67.5 min) and silybin B (tR = 74.5 min)
were collected, and after removal of the mobile phase under re-
duced pressure, amorphous colourless powders of silybin A
(300 mg) and silybin B (315 mg) were obtained by drying under
vacuum. To make the procedure quicker and to maximise the
functionality of our instruments, we carried out the injections in
considerably greater than in H2O or MeOH (0.05–1.5 mg/mL)
[20], allowing for the injection of large quantities of analyte. The
key point of our approach is dissolving the sample in THF, an al-
ternative solvent to H2O or MeOH in which silibinin has a very
low solubility. Various types of HPLC columns and conditions
(columns, flow, and mobile phases) were evaluated, and the opti-
mum chromatographic conditions for the separation of the two
diastereoisomers were determined by the use of a Phenomenex
Gemini C18-110A preparative column (10-µm particle size,
250 mm × 21.2 mm i.d.) with a mobile phase consisting of H2O/
MeOH/MeCN (60:35:5, v/v/v) and 0.1% TFA. In preliminary
studies, we also used DMSO, which dissolves silibinin very well
but resulted in poor resolution under all of the conditions tested.
To attain the highest quantity of pure silybin A and silybin B and
to avoid poor resolution due to the expansion of their chromato-
graphic peaks, 500-µL injections containing 70 mg of silibinin
were used. Better resolution was observed in all the tests per-
formed when the sample was dissolved in THF and mixed with
"
sequence every 40 min (l Fig. 3). Thus, we were able to obtain
approximately 1 g for each of the two diastereoisomers in a very
pure form in less than a week. The purified products, whose HPLC
"
purity was higher than 98% (l Fig. 4), were fully characterised by
NMR (1H, 13C), CD (Fig. 1S in Supporting Information), ESI MS, and
[α]D. The confirmation of two specific structures was also ob-
tained by comparing the NMR and CD data with those published
in the literature [3,4].
Afterwards, diastereoisomers 1A and 1B were treated with potas-
"
sium acetate in DMF at 80°C to produce 2A and 2B (l Fig. 1). In
"
an equal volume of the eluent solution. As shown in l Fig. 2, a
our procedure, a 0.1-M solution of pure silybin A (1A) or silybin
B (1B) was treated with anhydrous AcOK in DMF at 80°C for 2 h.
The crude mixture was purified by chromatography on a silica gel
good resolution was obtained when a 500-µL aliquot of 140 mg/
mL of the silibinin solution was mixed with 500 µL of the eluent
Di Fabio G et al. A Rapid and… Planta Med 2013; 79: 1077–1080

Letters 1079
Fig. 4 Analysis of silybin A (a) and silybin B (b) by
analytical HPLC: RP18 column Phenomenex LUNA
(5-µm particle size, 10.0 mm × 250 mm i.d.),
eluted with H2O/MeOH containing 0.01% acetic
acid (50:50, v/v), flow rate 0.8 mL/min, and moni-
tored at 288 nm.
Fig. 5 Analysis of 2,3-dihydrosilybin A (a) and 2,3-
dihydrosilybin B (b) by analytical HPLC: RP18 col-
umn Phenomenex LUNA (5-µm particle size,
10.0 mm × 250 mm i.d.), eluted with H2O/MeOH
containing 0.01% acetic acid (50:50, v/v), flow rate
0.8 mL/min, and monitored at 288 nm.
column (eluent: CHCl3/acetone, 80:20, v/v) to yield 2A and 2B in
good yields, and the HPLC purity was greater than 98% (l Fig. 5).
The purified products were fully characterised by NMR (1H, 13C),
CD, ESI MS, and [α]D. The obtained data were compared with
those published in the literature [12].
provides the opportunity to obtain large amounts of pure diaste-
reoisomers of silybin and enantiomers of 2,3-dehydrosilybin
with minimal effort, which opens up the possibility of conducting
further experiments to clarify the many biological properties of
these metabolites.
"
In conclusion, we have reported a simple and robust purification
method to obtain two diastereoisomers of silibinin in a very pure
form. Moreover, by starting with two separated diastereoiso-
mers, we have produced two enantiomers of 2,3-dehydrosilybin
in an optically pure form using our optimised oxidation proce-
dure. The described preparative HPLC method is effective in ob-
taining gram-scale amounts of the standards silybin A and silybin
B in a short period of time. All purified products were fully char-
acterised by NMR (1H, 13C), CD, [α]D, and ESI MS analyses. From
the HPLC analysis, the purity of the analogues obtained was de-
termined to be greater than 98% on average. Notably, this method
Material and Methods
!
The preparative HPLC method was performed with a Shimadzu
LC-8A PLC system equipped with a Shimadzu SCL-10A VP system
control and Shimadzu SPD-10A VP UV‑VIS detector. A Phenom-
enex Gemini C18-110A preparative column (10-µm particle size,
250 mm × 21.2 mm i.d.) was used, and the mobile phase of H2O/
MeOH/MeCN (60:35:5, v/v/v), containing 0.1% of TFA, was deliv-
ered isocratically at 12 mL/min. The chromatograms were moni-
tored at 288 nm. The HPLC system was controlled by LC Real-
Di Fabio G et al. A Rapid and… Planta Med 2013; 79: 1077–1080

1080 Letters
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Time Analysis software (Shimadzu Corporation). The silibinin so-
lution was prepared by dissolving and sonicating the accurately
weighed compound in THF. The obtained solution (ca. 140 mg/
mL) was then applied to Nylon filtres (pore size = 0.45 µm). A
500-µL volume of the silibinin solution was mixed with 500 µL
of the mobile phase and then applied to the chromatographic
system. Silybin A and silybin B peaks were collected manually.
The analysis was performed with a Waters 1525 HPLC equipped
with a binary gradient pump and a Waters 2996 Photodiode Ar-
ray Detector using an RP18 column Phenomenex LUNA (5-µm
particle size, 10.0 mm × 250 mm i.d.). The mobile phase of H2O/
MeOH, containing 0.01% acetic acid (50:50, v/v), was delivered
isocratically at 0.8 mL/min. The chromatograms were monitored
at 288 nm. Standard solutions of silybin A, silybin B, 2,3-dehydro-
silybin A, and 2,3-dehydrosilybin B were prepared by dissolving
the accurately weighed standard compounds in THF to yield final
concentrations of 5 mg/mL. A 25-µL volume of each analyte was
applied to the chromatographic system for the determination of
purity.
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Supporting information
Details on the general experimental procedures, the physico-
chemical data of compounds 1A, 1B, 2A, and 2B, the NMR data of
1A, 1B, and 2 as well as the CD spectra of compounds 1A, 1B, 2A,
and 2B are available as Supporting Information.
Acknowledgements
Financial support by AIPRAS Onlus (Associazione Italiana per la
Promozione delle Ricerche sullʼAmbiente e la Saluta umana) is ac-
knowledged. We also thank Tecno Bios® for the HPLC facilities.
Conflict of Interest
!
There are no conflicts of interest of all authors with respect to
this work.
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received February 11, 2013
revised
May 23, 2013
accepted May 24, 2013
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DOI http://dx.doi.org/10.1055/s-0032-1328703
Published online June 27, 2013
Planta Med 2013; 79: 1077–1080
© Georg Thieme Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
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Correspondence
Prof. Armando Zarrelli
Department of Chemical Sciences
University of Napoli ‘Federico II’
Via Cintia 4
80126 Napoli
Italy
Phone: + 39081674472
Fax: + 39081674393
zarrelli@unina.it
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