Acid-catalyzed epimerization of kobophenol A to carasinol B

Article abstract of DOI:10.3390/molecules13040938

The conversion from kobophenol A into carasinol B, two main chemical constituents of Caragana sinica, was confirmed by in vitro acid-catalyzed epimerization. The result provides important information about the biotransformation of kobophenol A in plants a

Full text of DOI:10.3390/molecules13040938

Molecules 2008, 13, 938-942
molecules
ISSN 1420-3049
© 2008 by MDPI
http://www.mdpi.org
Communication
Acid-catalyzed Epimerization of Kobophenol A to Carasinol B
Kejun Cheng, Gaolin Liang and Changqi Hu *
Department of Chemistry of Natural Drugs, School of Pharmacy, Fudan University, Shanghai 200032,
P. R. China; E-mails: chengkejun@gmail.com (for K. C.), stanfordliang@hotmail.com (for G. L.)
* Author to whom correspondence should be addressed; Tel: (+86)-21-54237564; Fax: (+86)-21-
64183411; E-mail: cqhu@shmu.edu.cn
Received: 13 March 2008; in revised form: 4 April 2008 / Accepted: 4 April 2008 / Published: 19
April 2008
Abstract: The conversion from kobophenol A into carasinol B, two main chemical
constituents of Caragana sinica, was confirmed by in vitro acid-catalyzed epimerization.
The result provides important information about the biotransformation of kobophenol A in
plants and its metabolism in rats.
Keywords: Kobophenol A; carasinol B; Caragana sinica.
Introduction
Caragana sinica (Buc’hoz) Rehd. (Fabaceae) is widely distributed in China. Its dried roots
(Chinese name: Jin-que-gen) have been used in China as a herbal medicine for the treatment of
asthenia syndrome, vascular hypertension, leukorrhagia, bruises and contused wounds. In a previous
study we found that the EtOAc extracts of the roots contained many oligostilbenes that had multi-
faceted bioactivities [1]. Kobophenol A (1), a stilbene tetramer, accounts for nearly 20% of the EtOAc
extracts. Using an improved E-screen assay, we found that 1 was a phytoestrogen with estrogenic
activity: it could compete with 17β-estradiol for binding to estrogen receptors, promote estrogen
receptor-mediated transcription and stimulate the growth of MCF-7 and osteoblasts [2]. During the
study on C. sinica, we found that the content of 1 decreased and that of other components increased
when the extracts were stored as an alcohol syrup. This instability caused great difficulties for the
quality control of this traditional Chinese herb medicine. The major component of the compounds
found in the increased quantities was a new stilbene tetramer, carasinol B (2), which was first isolated,

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Molecules 2008, 13
purified, and identified by us. As the isomer of 1, compound 2 also has stimulating activity on the
proliferation of osteoblasts [3]. Since the plant extracts are weakly acidic, we reckoned that an acidic
environment might be responsible for the observed transformation.
Further studies on the pharmacokinetics of 1 orally administered in rats showed that the absolute
oral bioavailability of 1 was 2.0%, and that 1 was mainly concentrated in the liver. These results
indicated that 1 should be metabolized either by liver microsomes or by gut microflora. Based on this,
we isolated new metabolites of 1: koboquinone A and koboquinone B, together with metabolite 2,
from rats’ feces [4]. In vitro incubation of 1 with intestinal bacteria indicated that the oxidative
metabolite koboquinone A was metabolized by the intestinal bacterium Klebsiella pneumoniae [5].
Probably some other intestinal bacterium produced the other oxidative metabolite koboquinone B.
However, we did not observe 2, the epimeric metabolite of 1, from intestinal bacteria. Results of these
experiments hint us that the epimeric metabolite 2 in rats might be the product of 1 by the action of
acidic gastric juice.
Results and Discussion
In a continuation of our efforts to study the chemical reactivity of 1 under acidic conditions,
different acids including inorganic acid, organic acid and Lewis acid were examined. The reactions
were monitored by HPLC analysis of total crude product mixtures obtained during each reaction.
Compound 2 was obtained when 1 was treated with three different acids. Among them, 0.01 M HCl
showed the best catalytic activity, while acetic acid with pH 5.0 and the Lewis acid BF3 displayed
weaker catalytic activities. The conversion rates of these reactions were 80%, 48% and 33%,
respectively. Benzylic carbons are reactive under acidic conditions. Recently Hatano et al. also
reported the acid catalyzed formation of an isomeric product of (-)-epigallocatechin gallate (EGCG)
[6]. In our study, ring C3 of 1 is a tetraphenol-substituted tetrahydrofuran moiety. The carbon atoms at
the 7c and 7d positions are the mostly probable to form tertiary carbocation transition states since they
are benzylic carbons and connected to an oxygen atom. Therefore, under acid catalysis, the bond
between atom C7c and atom O can break, and subsequently 2 forms (Figure 1).
Figure 1. Plausible mechanisms for the conversion of 1 to 2.
H+
H+
H
H
H
H
H
H
O
C3
Ф
Ф
O
C3
Ф
Ф
O
C3
Ф
Ф
7d
H 7c
(a)
H 7c
7d
H7c
7d
Ф
Ф
H
Ф
Ф
Ф
Ф
H
H
2
1

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Molecules 2008, 13
Ring A3 and ring B3 of 1 are also furan moieties, but they are benzofuran moieties. During the
experiments conducted, we did not obtain any products resulting from the epimerization of these two
rings. Based on the validation of the transformation above-mentioned, we suggest that 2 might be
partially biosynthesized from 1 in Caragana sinica and the transformation occurs when the extracts of
C. sinica are obtained as a weak acidic residue. Moreover, 1, ingested in animals, may be transformed
to 2 by the action of acidic gastric juice (Figure 2).
Figure 2. Conversion from 1 to 2.
HO
HO
H
4a
H
O
OH
HO
O
11b
OH
1a
7a
8a
13b
A3
A3
weak acidic extracts
HO
4c
10b
HO
HO
13a
9a
9b
8b
H
H
H
H
H
1c
7c
H
H
H
7c
H
H
11a
O
7b
O
C3
H
HCl or HOAc or BF3.OEt2
in rats
H
OH
1d
C3
OH
4d
1b
OH
8c
10c
B3
OH
7d
B3
7d
O
O
9c
8d
4b
11c
HO
H
HO
H
9d
13c
11d OH
OH
OH
OH
13d
HO
2
1
HO
Conclusions
A tetrastilbene carasinol B (2) has been obtained from its epimer kobophenol A (1). The
epimerization was catalyzed by different acids under mild conditions. The epimerization of 1 in vitro
supported the hypothesis that corresponding conversion would happen in the weak acidic extracts of
Caragana sinica and the acidic gastric juice when 1 were ingested by rats.
Experimental
General
UV spectra were obtained in absolute MeOH on a Shimadzu UV-260 spectrophotometer, IR
spectra on an Avatar 360 E.S.P Fourier Transform Infrared Spectrometer, and the optical rotation on a
1
13
JASCO P-1020 polarimeter in MeOH. H-NMR, C-NMR and 2D spectra were taken on a Mercury
Plus 400 NMR spectrometer in CD3OD. HRESIMS was recorded on a Q-Tof micro spectrometer.
Lobar RP-18 (40-63 µm, Merck) was used for column chromatography. An Agilent 1100 series
chromatography system with a photodiode array detector (DAD) and an ODS-2 Hypersil C18
reversed-phase column (5 μm, 15×0.46 cm) were used for HPLC analysis. Kobophenol A (1) was
isolated from the roots of Caragana sinica according to the literature [1].

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Molecules 2008, 13
General procedure for the formation of carasinol B (2) catalyzed by HCl or acetic acid
To a stirred solution of 1 (92.4 mg, 0.1 mmol) in methanol (3.0 mL) 0.01 M HCl (3.0 mL) (or 3.0
mL acetic acid with pH 5.0) was added at room temperature and the reaction mixture was refluxed for
18 hours. The solvent was removed under reduced pressure. The residue was then dissolved in
methanol/water (1/2, 1 mL) and applied to an ODS-2 RP-18 column to perform the column
chromatography. This afforded 73.9 mg of 2 (80%) as pale yellow amorphous powder (44.3 mg, 48%,
1
13
when acetic acid with pH 5.0 was used). All the product's data including [α], UV, IR, H-NMR, C-
NMR and HRESIMS agreed with the previous report [3].
General procedure for the formation of carasinol B (2) catalyzed by the Lewis acid BF3
To a stirred solution of 1 (92.4 mg, 0.1 mmol) in dried acetone (1.2 mL) 4 Å molecular sieves
(37.6 mg) was added at room temperature. After 10 minutes, boron trifluoride etherate (70 μL, 0.6
mmol) was added in dropwise fashion, and the reaction mixture was stirred at room temperature for 18
hours. The reaction mixture was filtered and the filtration was dried under reduced pressure. The
residue was then dissolved in methanol/water (1/2, 3 mL) under ultrasonication and then centrifuged.
The supernatant was applied to an ODS-2 RP-18 column to perform the column chromatography. This
afforded 30.5 mg of 2 (33%).
Figure 3. HPLC Chromatograms of 1 and 2.
kobophenol A (1)
9.886
mA
10
U
0
8
0
6
0
4
0
2
0
0
0
5
10
15
20
min
carasinol B (2)
5.101
mA
1
1
1
8
6
4
2
0
min
0
1
2
3
4
5
6
7
8
9

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Molecules 2008, 13
HPLC analysis of kobophenol A (1) and carasinol B (2)
The column temperature set at 30 °C. The mobile phase was a mixture of CH3CN/CH3OH/NaOAc-
HOAc buffer (pH 4.5, 0.02 M, 16:19:65, v/v/v) at a flow rate of 1.0 mL/min. The detection wave-
length was set at 284 nm. The injection volume was 20 μL. In the chromatograms of kobophenol A (1)
and carasinol B (2, Figure 3), the two compounds show obvious retention time differences and can be
easily distinguished.
Acknowledgements
This project is supported by the National Natural Science Foundation of China (No. 39270800)
and the Major Subject of the Science and Technology Commission of Shanghai Municipality (No.
06DZ19709). We are grateful to Professor Peng Xia of our University for his instruction in synthetic
experiments. Thanks also to Professor Caiyun Liu for her contributions to the NMR measurements.
References
1. Luo, H.F.; Zhang, L.P.; Hu, C.Q. Five novel oligostilbenes from the roots of Caragana sinica.
Tetrahedron 2001, 57, 4849-4854.
2. Tian, C.Y.; Hu, C.Q.; Xu, G.; Song, H.Y. Assessment of estrogenic activity of natural compounds
using improved E-screen assay. Acta. Pharmacol. Sin. 2002, 23, 572-576.
3. Ma, D.Y.; Luo, H.F.; Hu, C.Q. Three stilbene tetramers from the roots of Caragana sinica. Chin.
J. Chem. 2004, 22, 207-211.
4. Liang, G.L.; Cheng, K.J.; Zhou, H.; Hu, C.Q. Koboquinone A and B, new metabolites of
kobophenol A in rat. Chem. Pharm. Bull. 2004, 52, 1489-1491.
5. Liang, G.L.; Qian, L..S.; Cui, Y.L.; Hu, C.Q. Formation of oxidative metabolite from kobophenol
A by human intestinal bacterium Klebsiella pneumonia. Chem. Biodivers. 2005, 2, 506-509.
6. Hatano, T.; Hori, M.; Kusuda, M.; Ohyabu, T.; Ito, H.; Yoshida, T. Characterization of the
oxidation products of (-)-epigallocatechin gallate, a bioactive tea polyphenol, on incubation in
neutral solution. Heterocycles 2004, 63, 1547-1554.
Sample Availability: Samples of compounds 1 and 2 are available from the authors.
© 2008 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.