Biopreparation of an anti-inflammatory agent, diarctigenin, from arctiin isolated from Arctium lappa by Rhizoctonia solani AG-4

Article abstract of DOI:10.1016/j.tetlet.2013.10.057

In the preliminary screening for the plant-derived pesticides against Rhizoctonia solani Kühn AG-4 (RS AG-4), the indicator compounds arctiin (1) and arctigenin (2) in methanol extracts of Arctium lappa L. were consumed and transformed to other compounds. Thus, in the present study RS AG-4 was used as a biocatalyst and the biotransformation of arctiin (1) was investigated. Conversion of arctiin (1) to arctigenin (2) was achieved by the enzymatic hydrolysis of sugar moiety. In addition, an anti-inflammatory lignan dimer reported from the Arctium species, diarctigenin (3) was afforded in good yields. The HPLC monitoring of the biotransformation process indicated the possible mechanism. It would be an excellent method to produce large scale of diarctigenin (3) for the successive medicinal examinations.

Full text of DOI:10.1016/j.tetlet.2013.10.057

Tetrahedron Letters 54 (2013) 6955–6958
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Biopreparation of an anti-inflammatory agent, diarctigenin,
from arctiin isolated from Arctium lappa by Rhizoctonia solani AG-4
⇑
Ping-Chung Kuo , Zi-Yu Chen, Miao-Fan Chen
Department of Biotechnology, National Formosa University, Yunlin 632, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
In the preliminary screening for the plant-derived pesticides against Rhizoctonia solani Kühn AG-4 (RS
AG-4), the indicator compounds arctiin (1) and arctigenin (2) in methanol extracts of Arctium lappa L.
were consumed and transformed to other compounds. Thus, in the present study RS AG-4 was used as
a biocatalyst and the biotransformation of arctiin (1) was investigated. Conversion of arctiin (1) to arc-
tigenin (2) was achieved by the enzymatic hydrolysis of sugar moiety. In addition, an anti-inflammatory
lignan dimer reported from the Arctium species, diarctigenin (3) was afforded in good yields. The HPLC
monitoring of the biotransformation process indicated the possible mechanism. It would be an excellent
method to produce a large scale of diarctigenin (3) for the successive medicinal examinations.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 4 August 2013
Revised 30 August 2013
Accepted 14 October 2013
Available online 20 October 2013
Keywords:
Rhizoctonia
Arctium lappa
Arctiin
Diarctigenin
Biotransformation
Small organic molecules purified from natural sources provide an
incomparable source of inspiration for advances in organic chemis-
try and disease treatment due to their significant biological proper-
ties.1–7 Although some compounds can be easily synthesized by
chemical ways, some issues still remain, including complex opera-
tion, safety, pollution, and production cost. Biopreparation has been
considered to be an economical technology by synthetic organic
chemists in the development of new production routes for chemical
and pharmaceutical compounds. As compared with chemical syn-
thesis, biotransformation is a useful method to produce bioactive
compounds with advantages such as high stereo- and regio-selectiv-
ity, as well as milder reaction conditions, simple operation proce-
dures, and environmental safety.8–10
Arctium lappa L. is a perennial herb that has been cultivated as a
vegetable in many countries for a long time. The seeds of A. lappa L.
are extensively used in traditional medicines as diuretic, anti-
inflammatory, and detoxifying agents. The Arctium genus is a plen-
tiful source for the dibenzylbutyrolactone lignans, which are fa-
mous for their anti-proliferative and apoptotic effects. In the
previous literature, a dimeric lignan, diarctigenin (3), was reported
with significant inhibitory activity on nitric oxide (NO) production
in LPS-activated mouse peritoneal macrophages.11,12 Inflammation
is related to morbidity and mortality of many diseases and is
recognized as part of the complex biological response of vascular
tissues to harmful stimuli. It is the host response to infection or in-
jury, which involves the recruitment of leukocytes and the release
of inflammatory mediators, including NO. Sustained NO release by
inducible nitric oxide synthase (iNOS) and prostaglandin E2 (PGE2)
production by cyclooxy-genase 2 (COX2) have been implicated as
mediators of inflammation and are induced by bacterial lipopoly-
saccharide (LPS) or immunological stimuli. It has been reported
that excess production of NO and PGE2 by macrophages and other
cells exposed to endotoxins may contribute to septic shock, cere-
bral injury, myocardical ischemia, diabetes, arteriosclerosis, and
other local or systemic inflammatory disorders.13–16 Thus, inhibi-
tion of NO synthesis and PGE2 production stands as an important
therapeutic goal. Due to the low cytotoxicity of diarctigenin,12 it
could be a good candidate for the treatment of various anti-inflam-
matory diseases. However, the natural abundance of this dimeric
lignan is very limited for the successive bioactivity examinations.
O
7'
2'
H3CO
3'
9'
8'
1'
6'
O
4'
8
9
7
HO
5'
1
2
2
6
5
3
4
OCH3
OCH3
⇑
Corresponding author. Tel.: +886 5 6315491; fax: +886 5 6315502.
E-mail address: pcckuoo@nfu.edu.tw (P.-C. Kuo).
Figure 1. Chemical structure of diarctigenin (3) and significant HMBC (?)
correlations.
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.10.057

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P.-C. Kuo et al. / Tetrahedron Letters 54 (2013) 6955–6958
Figure 2. The HPLC chromatograms of the growth culture of RS AG-4 with arctiin (1) served as the starting substrate in the incubation period.
In addition, there was also no synthetic report regarding this com-
pound until now.
Rhizoctonia solani Kühn caused serious damping-off diseases of
numerous crops in Taiwan, especially it is a major problem for the
commercial production of vegetable seedlings grown in cell-plug
systems.17,18 In our labs, we continued to focus on the discovery
for the plant-derived pesticides against R. solani Kühn AG-4 (RS
AG-4).19 Among the tested extracts, the methanol extracts of seeds

P.-C. Kuo et al. / Tetrahedron Letters 54 (2013) 6955–6958
6957
RS AG-4, arctiin (1) was added into the medium and cultivated
for an additional 7 days, together with two controls, which con-
tained either mycelia with medium or substrate dissolved in DMSO
with medium. There were no metabolic products observed in two
control experiments. After the fermentation, the culture medium
and mycelia were filtered and fractionated by liquid–liquid parti-
tion to afford ethyl acetate and water soluble fractions, respec-
tively. With the assistance of a combination of conventional
chromatographic techniques, the ethyl acetate fractions afforded
two compounds, arctigenin (2) (21%) and diarctigenin (3) (37%).
The chemical structure of arctigenin (2) was characterized by com-
parison of its spectral and physical data with those reported in the
literature.11 Compound 3 was purified as optically active white
amorphous powder with mp 89–92 °C and ½a D25
ꢀ
–30 (c 0.1, CHCl3).
The positive-mode HR-ESI-MS of 3 showed a pseudomolecular ion
peak at m/z 765.2870 corresponding to a molecular formula of
C
42H46O12. The UV spectrum of compound 3 in methanol exhibited
Figure 3. The relative abundances of arctiin (1), arctigenin (2), and diarctigenin (3)
in the incubation period (n = 3–5).
characteristic absorption maxima of a lignan skeleton at 284 and
225 nm.20 The IR absorption bands at 3424, 1763, 1591 cmꢁ1 dis-
played the presence of hydroxyl, carbonyl groups, and carbon–car-
bon double bond, respectively. In the 1H NMR spectrum, a set of
three mutually coupled ABX-type proton signals at d 6.69 (1H, d,
J = 8.4 Hz, H-5), 6.53 (1H, dd, J = 8.4, 2.0 Hz, H-6), and 6.46 (1H, d,
J = 2.0 Hz, H-2), was indicative of a tri-substituted benzene ring.
Two mutually meta-coupled doublets at d 6.72 (1H, d, J = 2.0 Hz,
H-60) and 6.67 (1H, d, J = 2.0 Hz, H-20) were characteristic of a tet-
ra-substituted benzene ring. In addition, aliphatic methine and
methylene signals at d 4.14 (1H, m, H-9), 3.88 (1H, m, H-9), 2.96
(2H, d, J = 4.8 Hz, H-70), 2.69 (1H, m, H-7), and 2.56 (3H, m, H-7,
8, 80) were accounted for the butyrolactone-type lignan.11 These
spectral data were in accordance with the previously reported
of A. lappa L. (Arctii Fructus) did not show significant inhibition of
the growth of plant pathogenic fungi. However, the indicator com-
pound arctiin (1) in the methanol extracts of Arctii Fructus was
transformed to other compounds according to our preliminary
HPLC assays. Thus, in the present study the RS AG-4 was used as
a biocatalyst and the biotransformation of arctiin (1) was investi-
gated. In addition, the bioconversion was monitored by HPLC to
determine the relative abundance of each component involved in
the dynamic process.
Precultured RS AG-4 was transferred into a flask containing
medium and cultivated at 27 °C for 3 days. After the growth of
O
O
H
O
H
H3CO
H3CO
H3CO
O
O
O
protonation
O
GlcO
HO
RS AG-4
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
1a
1
2
O
O
O
H
H3CO
H3CO
O
H3CO
O
O
O
O
HO
dimerization
H+
H
H
2
2
aromatization
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
1c
1b
3
Figure 4. The plausible metabolic pathway for arctiin (1).

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P.-C. Kuo et al. / Tetrahedron Letters 54 (2013) 6955–6958
compound, diarctigenin (3).11 The full assignments of 1H and 13C
NMR signals were determined by the HMBC experiment (Fig. 1).
From the above spectral data, the chemical structure of com-
pound 3 was concluded to be diarctigenin. This is the first biosyn-
thetic Letter of diarctigenin and the present synthetic protocol
provides an efficient synthetic pathway with satisfied yield
(37%). In addition, the reagents used in this protocol were compar-
atively low cost, thus it would be suitable for the large-scale pro-
duction of diarctigenin.
In order to investigate the biopreparation mechanism, the time
course of biotransformation of arctiin (1) was analyzed by HPLC.
The optimized HPLC analytical conditions for the crude products
and the HPLC chromatograms of the indicator compounds arctiin
(1), arctigenin (2), and diarctigenin (3) were displayed in the Sup-
plementary data. The developed HPLC chromatographic analytical
method was applied to assess the relative abundances of the com-
pounds including arctiin (1), arctigenin (2), and diarctigenin (3) to
evaluate the changes occurred during the incubation. The typical
chromatograms of the crude products are displayed in Figure 2
and the relative abundances of arctiin (1), arctigenin (2), and diarc-
tigenin (3) in the incubation period are shown in Figure 3. The con-
tent of starting material arctiin (1) was decreased significantly in
the first 4 days; in contrast, arctigenin (2) and diarctigenin (3) were
produced and their contents were increased. Until 6 days the con-
tents of arctigenin (2) and diarctigenin (3) reached maxima and
would not increase any more.
In another HPLC assay, only arctigenin (2) served as the starting
substrate was incubated with the growth culture of RS AG-4, how-
ever, no metabolites of dimeric lignan diarctigenin (3) was found
(see the Supplementary data). It suggested that the formation of
diarctigenin should be related to the arctiin.
According to the results described above, the plausible meta-
bolic pathway for arctiin (1) was proposed as shown in Figure 4.
The glucose moiety connected with the lignan was hydrolyzed
and resulted in a phenolic radical (1a). This radical could capture
the hydrogen from the medium to afford the aglycone arctigenin
(2). Part of the intermediate radical was converted into the phe-
nylic radical (1b) and successive intermolecular dimerization
yielded dimeric intermediate product (1c). Finally the aromatiza-
tion of 1c furnished the diarctigenin (3). Without the presence of
arctiin (1), there is no accumulation of phenolic radical (1a) attrib-
uted to the formation of diarctigenin. Consequently, there is no
production of dimeric lignan diarctigenin.
In the present study RS AG-4 was used as a biocatalyst and arc-
tiin (1) was converted into arctigenin (2) and diarctigenin (3) both
in satisfied yields. The current Letter would be an excellent method
to produce a large scale of diarctigenin for the further medicinal
and bioactivity examinations.
Acknowledgment
Authors are grateful to the financial support of this research
from National Science Council, Taiwan, ROC.
Supplementary data
Supplementary data (experimental procedures, HPLC
chromatograms, and spectral data) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.tetlet.2013.10.057.
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