Biotransformation of Licochalcone A by transgenic crown galls of Panax quinquefolium

Article abstract of DOI:10.1016/j.molcatb.2014.09.015

Licochalcone A, as main constituent of Chinese Gancao (Glycyrrhiza inflate Bat), is responsible for thechemopreventive effect of the herbal. In order to get the bioactive modified molecules of Licochalcone A,it was biotransformed using transgenic crown galls of Panax quinquefolium to afford 9 products includinga new alkaloid together with 8 known compounds. Their structures were elucidated by physicochemicaland spectral methods as: methyl(12S)-1-[(5-formylfuran-2-yl)methyl]-5-oxopyrrolidine-2-carboxylate(1), methyl(12S)-1-[2-(furan-2-yl)-2-oxoethyl]-5-oxopyrrolidine-2-carboxylate (2), 5-hydroxymethyl-2-furfural (3), (S)-methylpyroglutamate (4), 4-hydroxy-2-methoxy-5-(2-methylbut-3-en-2-yl) benz-aldehyde (5), 4-hydroxybenzoicacid (6), dibutyl phthalate (7), 3β,6α,12β,25-tetrahydroxy-(20S,24R)-epoxy-dammarane (8), β-sitosterol (9).Among them, compounds 5 and 6 were transformed from Licochalcone A, while 14 and 8 were derivedfrom the culture medium stimulated by the substrate. Furthermore, their cancer chemopreventive effectwere tested by the assay of NQO1 (NAD(P)H: quinine oxidoreductase 1) inducing activities in Hepa 1c1ccells.

Full text of DOI:10.1016/j.molcatb.2014.09.015

Accepted Manuscript
Title: Biotransformation of Licochalcone A by Transgenic
Crown Galls of Panax quinquefolium
Author: Jiayuan Li Ning Li Bing Huang Weihong Zhao Wenli
Wang Yingying Si Chunyan Yan
PII:
S1381-1177(14)00260-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2014.09.015
MOLCAB 3036
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
27-8-2014
20-9-2014
24-9-2014
Please cite this article as: J. Li, N. Li, B. Huang, W. Zhao, W. Wang, Y.
Si, C. Yan, Biotransformation of Licochalcone A by Transgenic Crown Galls
of Panax quinquefolium, Journal of Molecular Catalysis B: Enzymatic (2014),
http://dx.doi.org/10.1016/j.molcatb.2014.09.015
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Graphical Abstract
To get more derivatives of Licochalcone A and study the ability of the transgenic crown galls of
Panax quinquefolium to transform natural chalcone, we modified the structure of Lic.A using
transgenic crown galls of P. quinquefolium. Nine products, including one new alkaloid, were
elucidated and the possible biosynthetic pathways were suggested. The toxicities and NQO1
inducing activities were assayed.
1
Page 1 of 22

Highlights
ꢀ
The transgenic crown galls of Panax quinquefolium broke the α/β unsaturated bond of
Licochalcone A.
ꢀ
ꢀ
ꢀ
New alkaloid was produced by the system with the stimulation of Licochalcone A.
The possible biosynthetic pathways were suggested.
The cytotoxicities and NQO1 inducing activities of products were assayed.
2
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Biotransformation of Licochalcone A by Transgenic Crown Galls
of Panax quinquefolium
a, b
Jiayuan Li a, b, Ning Li*a, b, Bing Huang c, Weihong Zhao a, b, Wenli Wang a, b, Yingying Si
,
Chunyan Yan *c,
a School of Traditional Chinese Materia Media, Shenyang Pharmaceutical University, Shenyang
110016, P.R. China
b
Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical
University), Ministry of Education, Wenhua Road 103, Shenyang 110016, China
c College of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, PR China
*To whom correspondence should be addressed. Tel: +86-24-23986475. Fax: +86-24-31509368.
E-mail address: liningsypharm@163.com
3
Page 3 of 22

Abstract:
Licochalcone A, as main constitutent of Chinese Gancao (Glycyrrhiza inflate Bat), is
responsible for the chemopreventive effect of the herbal. In order to get the bioactive
modified molecules of Licochalcone A, it was biotransformed using transgenic crown
galls of Panax quinquefolium to afford 9 products including a new alkaloid together
with 8 known compounds. Their structures were elucidated by physicochemical and
spectral methods as: methyl (12S)-1-[(5-formylfuran-2-yl) methyl]-5-oxopyrrolidine-
2-carboxylate (1), methyl (12S)-1-[2-(furan-2-yl)-2-oxoethyl]-5-oxopyrrolidine-
2-carboxylate (2), 5-hydroxymethyl-2-furfural (3), (S)-methylpyroglutamate (4),
4-hydroxy-2-methoxy-5-(2-methylbut-3-en-2-yl)
benzaldehyde
(5),
4-hydroxybenzoicacid (6), dibutyl phthalate (7), 3β, 6α, 12β, 25-tetrahydroxy-(20S,
24R)-epoxy-dammarane (8), β-sitosterol (9).
Among them, compounds 5 and 6 were transformed from Licochalcone A, while 1-4
and 8 were derived from the culture medium stimulated by the substrate. Furthermore,
their cancer chemopreventive effect were tested by the assay of NQO1 (NAD(P)H:
quinine oxidoreductase 1) inducing activities in Hepa 1c1c cells.
Keywords: licochalcone A; biotransformation; transgenic crown galls of Panax
quinquefolium; new alkaloid; NQO1, cancer chemoprevention.
1. Introduction:
Bioactive compounds in traditional species and herbs can protect and/or prevent
cancer development by inducing NQO1. These bioactive components are generally safe
and efficacious given that they have been consumed as medicine or food by humans for
4
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centuries. Licochalcone A (Lic.A; C21H22O4, MW 338, Fig.1) [1,2,3,4] is the main
active constituent of the licorice specie Glycyrrhiza inflate, which is also reported as an
estrogenic flavonoid with antitumor and antiparasitic properties. We previously
demonstrated that Lic.A might be the candidate cancer chemoprevention agent because
of the NQO1 inducing activity and reducing angiogenesis. In order to get more effective
NQO1 inducers and study the ability of P. quinquefolium to transform chalcones, we
modified the structure of Lic.A by biotransformation method.
The crown galls of transgenic Panax quinquefolium is the expressing production of
transgenic P. quinquefolium, in which the plant has been infected by Agrabacterium
tumefaciens, and the Ti plasmid in A. tumefaciens has been introduced into the plants’
cell nuclear genome [5,6]. The crown galls cultures, as a new form of culture system,
possesses distinct advantages when compared with callus and cell culture, such as faster
growth cycle, higher yield of transformed constituents, more secondary metabolites and
lower contamination of exogenous phytohormones [7] Thus, crown galls can be applied
to produce useful secondary metabolites efficiently and effectively.
Up to now, there is no report about biotransformation of Lic.A using the transgenic
crown galls of P. quinquefolium. Here, we focused on finding out novel bioactive
derived products of Lic.A by this special biotransformation system. As a result, one new
alkaloid together with 8 known products (Fig.1) was afforded from the suspension
cultures of transgenic crown galls of P. quinquefolium. The possible biosynthetic
pathways of compounds 1-9 were suggested. Furthermore the NQO1 inducing activities
were assayed using Hepa1c1c cells.
5
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O
OH
HO
O
substrate: Licochalcone A
transformed products
O
6
O
9
1
O
9
O
7
O
O
1
5
2
H
N
O
O
11
12
N
O
H
OH
6
5
3
4
11
2
12
7
O
4
13
3
O
O
3
14
14
2
1
COOH
O
H
O
OH
N
O
O
OHC
OH
6
4
5
HO
O
O
OH
O
O
O
HO
HO
OH
8
7
9
Fig. 1. stucture of compounds 1-9
O
O
O
H
N
O
O
Fig. 2. Key HMBCs(H-C ) of compound 1
2. Material and methods
2.1 General
NMR spectra: Bruker ARX-300 or ARX-600 spectrometer was used, with TMS as an
internal standard. ESI-MS: Finnigan LCQ mass spectrometer was used. HR-ESI-MS:
6
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Bruker micro TOF-Q mass spectrometer was used to collect data; in m/z (rel.%). Silica
gel for chromatography was produced by Qingdao Ocean Chemical Group Co. of
China. HPLC separation was performed on YMC-pack PREP-ODS column
(250×20mm) equipped with Shimadzu RID-20AUV detector and a Shimadzu LC-6AD
series pumping system. Dimethyl sulfoxide (DMSO), thiazolyl blue (MTT) CDCl3,
DMSO-d6 and Pyridine-d5 were obtained from Sigma-Aldrich Company (St. Louis,
MO, USA). Minimum Essential Medium Alpha Medium (α-MEM), Fetal Bovine
Serum (FBS) were purchased from Invitrogen Corporation (New York, USA).
2.2 Plant material
The root of Glycyrrhiza inflata Batal. was collected in XinJiang Uighur Autonomous
Region, China, in August, 2010. The plant material was identified by Prof. Xiaoguang
Jia. (Xinjiang Institute of Chinese Material Medica and Ethnodrug). Avoucher specimen
(No.20100811) is deposited in Xinjiang Institute of Chinese Material Medica and
Ethnodrug.
2.3 Extraction and isolation
2.3.1: methyl (12S)-1-[(5-formylfuran-2-yl)methyl]-5-oxopyrrolidine-2-carboxylate (1).
1
Yellow oil,
-13.2 (c 0.55, MeOH). H-NMR and 13C-NMR see Table 1;
HR-ESI-MS (m/z 274.0687 ([M+Na+]; cal.C12H13NO5, 274.0680)). (Table 1)
2.3.2: methyl (12S)-1-[2-(furan-2-yl)-2-oxoethyl]-5-oxopyrrolidine-2-carboxylate (2)
Yellow powder.
-23.0 (c 1.0, MeOH). 1H-NMR and 13C-NMR see Table 1. [8,
9]
2.3.3: 5-hydroxymethyl-2-furfural (3)
7
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1
Light yellow oil-like solid. H-NMR (DMSO-d6, 600MHz): δ 9.53 (1H, s, -CHO),
7.48 (1H, d, J = 2.6 Hz, H-3), 6.59 (1H, d, J = 2.6 Hz, H-4), 5.55 (1H, t, -OH), 4.49
13
(2H, d, 7-CH2). C-NMR (DMSO-d6, 125MHz): δ 178.0 (-CHO), 162.2 (C-5), 151.8
(C-2), 124.4 (C-3), 109.8 (C-4), 56.0 (-CH2). [10]
2.3.4: (S)-methylpyroglutamate (4)
1
White amorphous powder,
-0.5 (c 0.75, MeOH). H-NMR (DMSO-d6, 400
MHz): δ 7.99 (s, 1H, -NH), 4.18 (dd, J = 9.1, 4.2 Hz, H-2), 3.67(s, -OCH3), 2.37-2.29
(m, H-4a), 2.16 (m, H-4b), 2.13 (m, H-3), 1.98 (m, H-3). 13C-NMR (DMSO-d6,
100MHz): δ 177.1 (C-5), 173.4 (COOCH3), 54.7 (-OCH3), 52.1 (C-2), 28.9 (C-5), 24.5
(C-4). [11]
2.3.5: 4-hydroxy-2-methoxy-5-(2-methylbut-3-en-2-yl)benzaldehyde (5)
1
Yellow oil, H-NMR (600MHz, DMSO-d6): δ 10.11 (1H, s, -CHO), 7.53 (1H, s,
H-6), 6.54 (1H, s, H-3), 6.16 (1H, dd, J = 10.7, 17.5 Hz, H-2'), 4.94 (2H, m, H-3'), 1.39
(6H, s, H-4', 5'), 3.81 (3H, s, 4-OCH3). 13C-NMR (125MHz, DMSO-d6): δ 186.7 (C=O),
162.0 (C-2, 4), 126.9 (C-5, 6), 115.9 (C-1), 99.6 C-3), 55.5 (-OCH3), 147.2 (C-2'), 110.4
(C-3'), 26.6 (C-4", 5"). [12]
2.3.6: 4-hydroxybenzoic acid (6)
1
Yellow oil, H-NMR (400MHz, DMSO-d6): δ 12.38 (1H, s, -COOH), 10.20 (1H, s,
-OH ), 7.78 (2H, d, H-3, 5, J = 8.6HZ), 6.81 (2H, d, H-2, 6, J = 8.6HZ). [12]
2.3.7: dibutylphthalate (7)
1
Light yellow oil, H-NMR (DMSO-d6, 400MHz): δ 7.72 (2H, s, H-2, 4), 7.70-7.65
(m, 2H, H-3, 5), 4.22 (4H, t, J = 6.5 Hz, H-8, 8'), 1.68-1.60 (4H, m, H-9, 9'), 1.37 (4H,
8
Page 8 of 22

m, H-10, 10'), 0.91 (6H, t, J = 7.6Hz, H-11, 11').[13]
2.3.8: 3β, 6α, 12β, 25-tetrahydroxy-(20S, 24R)-epoxy-dammarane (8)
1
Yellow solid, H-NMR (400MHz, CDCl3): δ 4.11 (m, 1H, H-4), 3.84 (dd, 1H, J =
8.2, 7.0 Hz), 3.51 (m, 1H, H-12), 3.17 (dd, 1H, J= 11.1,3.7 Hz), 1.31 (s, 3H, H-28),
1.27 (s, 3H, H-29), 1.27 (s, 3H, H-27), 1.09 (s, 3H, H-21), 1.06 (s, 3H, H-26), 0.97 (s,
3H, H-18 ), 0.93 (s, 3H, H-19), 0.91 (s, 3H, H-30). 13C-NMR (100MHz, CDCl3): δ 39.2
(C-1), 28.0 (C-2), 78.7 (C-3), 39.4 (C-4), 61.4 (C-5), 68.8 (C-6), 47.1 (C-7), 41.0 (C-8),
50.1 (C-9), 38.9 (C-10), 31.3 (C-11), 71.0 (C-12), 49.1 (C-13), 52.0 (C-14), 32.7 (C-15),
25.1 (C-16), 48.0 (C-17), 17.1 (C-18), 17.5 (C-19), 86.6 (C-20), 26.3 (C-21), 31.0
(C-22), 28.7 (C-23), 85.7 (C-24), 70.2 (C-25), 27.2 (C-26), 27.7 (C-27), 31.3 (C-28),
15.6 (C-29) , 18.3 (C-30).[14]
2.3.9: Licochalcone A
1H-NMR (600MHz, DMSO-d6): δ 7.98 (2H, d, J = 8.9 Hz, H-2', 6'), 6.89 (2H, d, J =
8.9 Hz, H-3', 5'), 7.90 (1H, d, J = 15.6 Hz, H-β), 7.63 (1H, d, J = 15.6 Hz, H-α), 7.53
(1H, s, H-6), 6.53 (1H, s, H-3), 6.25 (1H, dd, J = 10.3, 17.8 Hz, H-2"), 4.96 (1H, m,
13
H-3"a), 4.91 (1H, m, H-3"b), 1.46 (6H, s, H-4", 5"), 3.83 (3H, s, 2-OCH3). C- NMR
(125MHz, DMSO-d6): δ 187.4 (C=O), 161.9 (C-4'), 159.9 (C-4), 158.4 (C-2), 147.6
(C-2"), 138.8 (C-β), 130.8 (C-2', 6'), 129.7 (C-1'), 127.8 (C-6), 126.7 (C-5), 117.8
(C-α), 115.4 (C-3', 5'), 113.6 (C-1), 110.1 (C-3"), 100.1 (C-3), 55.5 (-OCH3), 40.4
(C-1"), 27.1 (C-4"), 27.1 (C-5"). [12]
2.4 Methods
2.4.1 Biotransformation of Licochalcone A by suspension cultures of transgenic crown
9
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galls of Panax quinquefolium
The crown galls were induced from the direct infection of P. quinquefolium sterile
stems with A. tumefaciens C58, and then were cultured in the MS liquid medium (3%
(w/v) sucrose) without phytohormones. The crown galls were sub-cultured into a fresh
medium at 30 day intervals.
The pH value of the medium was adjusted to 5.75 before sterilization. 10 g fresh
transgenic crown galls were inoculated into a 500 ml Erlenmeyer flask with 200 ml MS
medium, which were cultured at 25 °C on an orbital shaker at 110 rpm in the darkness.
[15]
Licochalcone A (8mg) was dissolved in methanol and distributed into the suspension
cultures of crown galls of P. quinquefolium, there were in total of 115 bottles of the
same. The mixture was co-cultured for seven days, and then the cultures and the
medium were separated by filtration. The medium was extracted by EtOAc and
n-BuOH successively to get Fr.1 (EtOAc extract) (10g) and Fr.2 (n-BuOH extract) (4g).
The culture was dried under 55℃and crushed, then soaking with methanol for 12 h and
ultrasonic extraction for 45 minutes. The methanol extract was partitioned by EtOAc
and n-BuOH respectively to afford Fr.3 (EtOAc extract) (3.6g) and Fr.4 (n-BuOH
extract) (13.9g). Compared with the experimental group, culture without Lic. A act as
control and was processed to get Fr.5-8 using the same method.
2.4.2 Purification of biotransformation products
Fr.1 (10g) was chromatographied on ODS column eluted by MeOH/H2O (from 100:0
to 0:100) to obtain eleven fraction (Fr.1a-11a). Fr.2a (10:90) was separated by
10
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preparative TLC and the eluent was petroleum : EtOAc=2:1 to afford subfraction 2a-1,
2a-2 and 2a-3. Then subfraction 2a-1 was purified by RP-HPLC with a ODS column
(250×10 mm, flow rate 2ml/min) with MeOH/H2O (60:40) in 17min to afford
compound 4 (4.5mg) and in 19min to get compound 7 (3.8mg). While subfraction 2a-2
was purified by RP-HPLC with a ODS column (250×10 mm, flow rate 2ml/min) with
MeOH/H2O (45:55) in 30min to obtain Compound 2 (1.5mg) and in 38min to purify
compound 1 (4.5mg). Compound 5 (4.0mg) was obtained from subfraction 2a-3 by
RP-HPLC with a ODS column (250×10 mm, flow rate 2ml/min) with MeOH/H2O
(55:45) in 20min. Fr.7 (60:40) was separated by Sephadex LH-20 chromatography
eluted with MeOH to afford subfraction 7-1, 7-2. Furthermore, compound 3 (1.8mg)
was isolated from subfraction 7-1 using preparative RP-HPLC with a ODS column
(250×10 mm, flow rate 2ml/min) with MeOH/H2O (60:40) in 50min. Compound 6
(7.4mg) was obtained from subfraction 7-2 using preparative RP-HPLC with a ODS
column (250×10 mm, flow rate 2ml/min) with MeOH/H2O (65:35) in 45 min, and
purified by recrystalization in MeOH. Moreover, Fr.11 was separated with silica gel
column chromatography (PE/EtOAc 100:0 to 0:100) and then purified by
recrystalization to afford compound 9 (13.4mg). Fr.9 was isolated using preparative
HPLC with MeOH/H2O (80:20) in 34min and purified by Sephadex LH-20
chromatography eluted with MeOH to yield compound 7 (1.4mg). Fr.4 (5g) was
chromatographied on ODS column eluted by MeOH/H2O (from 100:0 to 0:100) to
obtain eleven fraction (Fr.1b-11b). Compound 8 is from the Fr.1b (10:90) using
preparative RP-HPLC with a ODS column (250×10 mm, flow rate 2ml/min) with
11
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MeOH/H2O (55:45) in 35 min.
2.4.3 NAD(P)H: quinine oxidoreductase 1 inducing activities assay
Activity of NQO1 was determined in Hepa 1c1c7 cells as described in the previous
study [16, 17, 18]. Cells (104cells/well) were incubated in 96-well plate for 24 h after
administration. The media was removed and cells were lysed by a solution (50uL)
containing 0.8% (w/v) digitonin and 2 mM EDTA under agitation for 10 min. Then
200μL of freshly prepared reaction solution (40 mL of reaction solution consisted of 2
mL of 0.5 M Tris–HCl (pH 7.4), 0.27 mL of 1.5% Tween-20, 0.027 mL of 7.5 mM
flavin adenine dinucletide, 0.27 mL of 150 mM glucose-6-phosphate, 0.024 mL of 50
mM NADP, 26.7 mg of bovine serum albumin, 12 mg of MTT, 0.04 mL of 50 mM
menadione, 80 units of yeast glucose-6-phosphate dehydrogenase and deionized water
was added to reach the final volume) was added into every well Five minutes later, the
plate was read at 550 nm. Stock solutions of the eight compounds were dissolved in
DMSO, and the final concentration of DMSO was kept at 0.5% (v/v). 4'-Bromoflavone
in DMSO (final concentration: 4μM) and DMSO were used as the positive and negative
control, respectively, and their final concentration of DMSO were both kept at 0.5%
(v/v). IR (fold of NQO1 induction compared to negative levels) values were considered
as the records of the NQO1 induction activity.
3. Results and discussion
Spectra (1D and 2D NMR, MS, [α]D) analysis revealed the structures of isolated
products as: methyl (12S) -1-[(5-formylfuran-2-yl) methyl]-5-oxopyrrolidine-2-
carboxylate (1), methyl (12S) -1- [2-(furan-2-yl)-2-oxoethyl]-5-oxopyrrolidine-2-
12
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carboxylate (2), 5-hydroxymethyl-2-furfural (3), (S)-methylpyroglutamate (4),
4-hydroxy-2-methoxy-5-(2-methylbut-3-en-2-yl) benzaldehyde (5),
4-hydroxybenzoicacid (6), dibutyl phthalate (7), 3β, 6α, 12β, 25-tetrahydroxy-(20S,
24R)-epoxy-dammarane (8), β-sitosterol (9). Among them, 1 was a new compound.
Compound 1 was obtained as yellow oil,
-13.2 (c 0.55, MeOH), with a
molecular formula of C12H13NO5 determined by HR-ESI-MS ion at m/z 274.0687 (cal.
274.0680 for C12H13NO5Na), which indicated 7 degrees of unstauration. The 1H and 13C
NMR data indicated characteristic signals for a furan ring at δ 7.49 (1H, d, J = 3.5 Hz,
H-4) and δ 6.63 (1H, d, J = 3.5 Hz, H-3). The signals for methoxy group and aldehyde
group were observed at δ 3.63 (3H, s, 14-OCH3) and δ 9.53 (1H, s, 6-CHO). The
resonances at δ 1.95 - 2.01 (1H, m, H-11a) and 2.28 - 2.36 (3H, m, H-11b, H-10) were
attributed to the methylene groups respectively. In the 13C NMR spectra, 12 carbon
signals were afforded accordingly including 3 carbonyl groups. The lower-field shift
observed as δ 38.2 (C-7) and δ 58.8 (C-12) revealed that they were connected to the
N-atom. Furthermore the long-range correlations shown in HMBC spectra (Fig.2)
between δ 4.31 (H-7a), δ 4.71 (H-7b) and δ 58.8 (C-12), 174.5 (C-9), 156.5 (C-2)
confirmed this connection. While the HMBC correlations between δ 9.53 (-CHO) and
152.1 (C-5), δ 4.24-4.22 (H-12) and 172.5 (C-13) were also observed, which suggested
a linkage of C-6 to C-5 and C-14 to C-12. Thus, compound 1 was determined as
methyl-1-[(5-formylfuran-2-yl) methyl]-5-oxopyrrolidine-2-carboxylate.
There are similar structure skeleton in compounds 1 and 2, with different location of
carbonyl group. So the absolute configuration of the C-12, the chiral center in
13
Page 13 of 22

compound 1, was elucidated by comparing the data of
(-13.2) with that of
compound 2 (-23.0) reported [8, 9]. Then, the absolute configuration of C-12 was
determined as S-configuration. Above all, the compound was identified as methyl (12S)
-1-[(5-formylfuran-2-yl) methyl]-5-oxopyrrolidine-2-carboxylate.
Table 1 NMR data of compound 1 and 2 (1H: 600 MHz, 13C: 150 MHz in DMSO-d6)
1
2
Position
C
H
C
H
2
3
4
5
6
156.5
111.5
124.1
152.1
178.0
150.2
119.0
112.6
148.1
182.7
7.48 (1H, d, J = 3.5 Hz)
6.62 (1H, d, J = 3.5 Hz)
7.57 (1H, d, J = 3.5 Hz)
6.74-6.75 (1H, d, J = 3.5 Hz)
8.04 (s)
9.53 (s)
4.71 (1H, d, J = 18.0 Hz)
4.31 (1H, d, J = 18.0 Hz)
4.84 (1H, d, J = 18.0 Hz)
4.34 (1H, d, J = 18.0 Hz)
7
38.2
47.3
9
174.5
28.6
174.9
28.5
10
2.36-2.28 (m)
2.36-2.30 (m)
2.36-2.28 (m)
2.01-1.95 (m)
2.46-2.37 (m)
2.07-2.01 (m)
11
22.5
22.3
58.8
172.5
52.3
4.24-4.22 (m)
59.4
172.1
52.1
4.35-4.33 (m)
12
13
15
3.63 (s)
3.75 (s)
Base on the structures of transformed products, the possible biosynthesis pathway
was supposed. Firstly, Lic. A was transformed into compound 5 (4-hydroxy-2-methoxy
-5-(2-methylbut-3-en-2-yl) benzaldehyde) and 6 (4-hydroxybenzoic acid) through
rupturing the α/β unsaturated bond of Lic.A as shown in Fig 3. While, compounds 1-4
and 8 were derived from the culture medium stimulated by Lic. A. It is the first time to
identify compounds 1-4 and 8 from the system of P. quinquefolium. Both compounds 3
and 4 were derived from glucose through the specific process described in Fig 4. Then,
compound 1 was constructed by compounds 3 and 4 through condensation reaction
14
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between the hydroxyl group in 2 and amino group in 4. With similar pathway,
compound 2 came from compounds 4 and 3a, an isomer of 3, as shown in Fig.4.
Compound 8 was from ocotillol-type triterpenes glycoside of the system of P.
quiquefolium itself deglycosylating.
O
OH
COOH
HO
O
OH
+
OHC
O
OH
4
Licochalcone A
3
Fig. 3. Possible biosynthesis pathway of compounds 5 and 6 from Lic. A in transgenic
crown galls of Panax quinquefolium
CH2OH
O
O
CH2OH
OH
O
HOH2C
O
HOH2C
O
O
OHC
HO
OHC
CH2OH
CHO
-H2O
N
OH
OH
-H2O
condensation
O
CHOH
OH
HO
HO
OH
3
CHO
OH
CH2OH
O
HO
HO
1
O
OH
OH
OH
OH
N
COOCH3
H
4
CH2OH
CH2OH
O
OH
CH2OH
OH
O
condensation
Glucose
OH
O
O
OH
O
OH
N
O
O
O
OH
-H2O
OH
OH
O
O
-H2O
OH
OH
-H2O
HO
OH
O
tricarboxylic
OH
3a
acid cycle
2
CH2OH
O
methylation
COOH
oxidation
hydroxylation
N
COOCH3
HO
2-Ketoglutaric acid
H
N
COOCH3
N
N
H
COOCH3
H
H
4
Fig. 4. Possible biosynthesis pathway of compounds 1-4 in transgenic crown galls of
panax quinquefolium stimulated by Lic.A
15
Page 15 of 22

4
3
2
1
0
1.5
1.0
0.5
0.0
0
Lic.A
1
2
3
4
5
6
7
0
Lic.A
1
2
3
4
5
6
7
Fig. 5. The cytotoxicities and NQO1 inducing activities of compounds 1-7 (at 20 μM)
(0: 4'-bromoflavone- positive control drug)
In order to evaluate the chemopreventive effect of the products we got, the
cytotoxicities and NQO1 inducing activities were tested respectively using Hepa 1c1c7
cells. Considering the assay results, it’s shown that all the tested compounds (20μM)
exhibited lower cytotoxicities than that of the substrate, Lic.A. And compared with
Lic.A, the NQO1 inducing activities of all the transformed products reduced to varying
degrees. Among them, compounds 1, 2 and 5, with IR value 1.69, 1.70, 1.65
respectively, exhibited better activities than those of compounds 4, 6 and 7 with IR
value 1.11, 1.20, 1.20.
As shown in Fig 5, the transformed products from Lic.A (compounds 5 and 6)
exhibited lower toxicities and NQO1 inducing activities comparing with Lic.A. One
possible reason for the decreasing of bioactivities may be the rupturing of the α、β
unsaturated bond of Lic.A, which is the potential active position for NQO1 inducing
activities.
4. Conclusions
Usually, the system of transgenic crown galls of panax quinquefolium was reported to
use for the glycosylation of substrate, including the biotransformation of phenolic acids
16
Page 16 of 22

[19], anthraquinone [20], sesquiterpenes [21]. For example, in the biotransformation
system of crown galls of P. quinquefolium: paeonol was monoglucosylated to yield
paeonol-2-O-β-D-glucopyranoside [20]. While, in our research, it’s the first time to
biotransform chalcone using the transgenic crown galls of P. quinquefolium. Lic.A, a
natural chalcone, was biotransformed by rupturing the bond of α/β unsaturated ketone
but not being glycosylated. Moreover, new compounds were produced by the system
with the stimulation of Lic.A and the biosynthesis pathway was also suggested.
Acknownlegements
The work was supported partially by National Natural Science Foundation of China
(81173531 and 81473108), Training Program Foundation for the Distinguished Young
Scholars of University in Liaoning Province (LJQ2012088), Fund for long-term
training of young teachers in Shenyang Pharmaceutical University (ZCJJ2013409),
Scientific Research Foundation for the Returned Overseas Scholars in Shenyang
Pharmaceutical University (GGJJ2013103).
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H. Matsuura, J. Agric. Food. Chem. 61 (2013) 9155-9159.
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19
Page 19 of 22

Table 1 NMR data of compound 1 and 2 (1H: 600 MHz, 13C: 150 MHz in DMSO-d6)
1
2
Position
C
H
C
H
2
3
4
5
6
156.5
111.5
124.1
152.1
178.0
150.2
119.0
112.6
148.1
182.7
7.48 (1H, d, J = 3.5 Hz)
6.62 (1H, d, J = 3.5 Hz)
7.57 (1H, d, J = 3.5 Hz)
6.74-6.75 (1H, d, J = 3.5 Hz)
8.04 (s)
9.53 (s)
4.71 (1H, d, J = 18.0 Hz)
4.31 (1H, d, J = 18.0 Hz)
4.84 (1H, d, J = 18.0 Hz)
4.34 (1H, d, J = 18.0 Hz)
7
38.2
47.3
9
174.5
28.6
174.9
28.5
10
2.36-2.28 (m)
2.36-2.30 (m)
2.36-2.28 (m)
2.01-1.95 (m)
2.46-2.37 (m)
2.07-2.01 (m)
11
22.5
22.3
58.8
172.5
52.3
4.24-4.22 (m)
59.4
172.1
52.1
4.35-4.33 (m)
12
13
15
3.63 (s)
3.75 (s)
20
Page 20 of 22

O
OH
HO
O
substrate: Licochalcone A
transformed products
O
6
O
9
1
O
9
O
7
O
O
1
5
2
H
N
O
O
11
12
N
O
H
OH
6
5
3
4
11
2
12
7
O
4
13
3
O
O
3
14
14
2
1
COOH
O
H
O
OH
N
O
O
OHC
OH
6
4
5
HO
O
O
OH
O
O
O
HO
HO
OH
8
7
9
Fig. 1. stucture of compounds 1-9
O
O
O
H
N
O
O
Fig. 2. Key HMBCs(H-C ) of compound 1
21
Page 21 of 22

O
OH
COOH
HO
O
OH
+
OHC
O
OH
4
Licochalcone A
3
Fig. 3. Possible biosynthesis pathway of compounds 5 and 6 from Lic. A in transgenic
crown galls of Panax quinquefolium
CH2OH
O
O
CH2OH
OH
O
HOH2C
O
HOH2C
O
O
OHC
HO
OHC
CH2OH
CHO
-H2O
N
OH
OH
-H2O
condensation
O
CHOH
OH
HO
HO
OH
3
CHO
OH
CH2OH
O
HO
HO
1
O
OH
OH
OH
OH
N
COOCH3
H
4
CH2OH
CH2OH
O
OH
CH2OH
OH
O
condensation
Glucose
OH
O
O
OH
O
OH
N
O
O
O
OH
-H2O
OH
OH
O
O
-H2O
OH
OH
-H2O
HO
OH
O
tricarboxylic
OH
3a
acid cycle
2
CH2OH
O
methylation
COOH
oxidation
hydroxylation
N
COOCH3
HO
2-Ketoglutaric acid
H
N
COOCH3
N
N
H
COOCH3
H
H
4
Fig. 4. Possible biosynthesis pathway of compounds 1-4 in transgenic crown galls of
panax quinquefolium stimulated by Lic.A
1.5
4
3
1.0
2
0.5
1
0.0
0
0
Lic.A
1
2
3
4
5
6
7
0
Lic.A
1
2
3
4
5
6
7
Fig. 5. The cytotoxicities and NQO1 inducing activities of compounds 1-7 (at 20 μM)
(0: 4'-bromoflavone- positive control drug)
22
Page 22 of 22