The comparison of neuroprotective effects of isoliquiritigenin and its Phase I metabolites against glutamate-induced HT22 cell death

Article abstract of DOI:10.1016/j.bmcl.2016.10.072

It is becoming increasingly important to investigate drug metabolites to evaluate their toxic or preventive effects after administration of the parent compound. In our previous study, isoliquiritigenin isolated from Glycyrrhizae Radix effectively protected mouse-derived hippocampal neuronal cells (HT22) against 5 mM glutamate-induced oxidative stress. However, there is little information on the protective effects of the metabolites of isoliquiritigenin on HT22 cells. In this study, isoliquiritigenin and its Phase I metabolites were prepared and their neuroprotective activities on glutamate-treated HT22 cells were compared. The prepared metabolites were liquiritigenin (1), 2′,4,4′,5′-tetrahydroxychalcone (2), sulfuretin (3), butein (4), davidigenin (5), and cis-6,4′-dihydroxyaurone (6). Among the six metabolites, 4 showed better neuroprotective effects than the parent compound, isoliquiritigenin. Our study suggests that the neuroprotective effect of isoliquiritigenin could be elevated by its active metabolite 4, which is a chalcone containing a catechol group in the B ring.

Full text of DOI:10.1016/j.bmcl.2016.10.072

Accepted Manuscript
The comparison of neuroprotective effects of isoliquiritigenin and its Phase I
metabolites against glutamate-induced HT22 cell death
Eun-Ju Yang, Minjun Kim, Ji Eun Woo, Taeho Lee, Jong-Wha Jung, Kyung-
Sik Song
PII:
S0960-894X(16)31110-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.10.072
BMCL 24375
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 August 2016
3 October 2016
24 October 2016
Please cite this article as: Yang, E-J., Kim, M., Eun Woo, J., Lee, T., Jung, J-W., Song, K-S., The comparison of
neuroprotective effects of isoliquiritigenin and its Phase I metabolites against glutamate-induced HT22 cell death,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.10.072
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The comparison of neuroprotective effects of isoliquiritigenin and its Phase I metabolites
against glutamate-induced HT22 cell death
Eun-Ju Yanga,†, Minjun Kima,†, Ji Eun Woob, Taeho Leea, Jong-Wha Junga,*, Kyung-Sik Songa,b,*
aResearch Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National
University, 80 Daehak-ro, Sankyuk-dong, Daegu 41566, Republic of Korea
bGham BioPharm, Co. Ltd., #401, College of Pharmacy, Kyungpook National University, 80
Daehak-ro, Sankyuk-dong, Daegu 41566, Republic of Korea
† These two authors contributed equally to this work.
* Corresponding authors. Tel.: +82 53 950 8565; fax: +82 53 950 8557.
E-mail address:kssong@knu.ac.kr (K.-S. Song), jungj@knu.ac.kr (J.-W. Jung).
1

ABSTRACT
It is becoming increasingly important to investigate drug metabolites to evaluate their toxic or
preventive effects after administration of the parent compound. In our previous study,
isoliquiritigenin isolated from Glycyrrhizae Radix effectively protected mouse-derived
hippocampal neuronal cells (HT22) against 5 mM glutamate-induced oxidative stress. However,
there is little information on the protective effects of the metabolites of isoliquiritigenin on HT22
cells. In this study, isoliquiritigenin and its Phase I metabolites were prepared and their
neuroprotective activities on glutamate-treated HT22 cells were compared. The prepared
metabolites were liquiritigenin (1), 2,4,4,5-tetrahydroxychalcone (2), sulfuretin (3), butein (4),
davidigenin (5), and cis-6,4-dihydroxyaurone (6). Among the six metabolites, 4 showed better
neuroprotective effects than the parent compound, isoliquiritigenin. Our study suggests that the
neuroprotective effect of isoliquiritigenin could be elevated by its active metabolite 4, which is a
chalcone containing a catechol group in the B ring.
Keywords:
Isoliquiritigenin; Phase I metabolites; Butein; Neuroprotection; HT22; Glutamate
2

Isoliquiritigenin (ISOLIQ), a kind of chalcone, is known as a key component of
Glycyrrhizae Radix (licorice roots),1-3 and has various biological effects such as anti-cancer,4-5
anti-inflammatory,6 and neuroprotective1-3 effects. By inducing mitochondrial dysfunction,
ISOLIQ promotes apoptosis of the prostate cancer cells, MAT-LyLu and DU145, which are
derived from rat and human, respectively.5 It also induces heme-oxygenase (HO)-1 expression
by activating extracellular signal-regulated kinases (ERK) 1/2, which are associated with
lipopolysaccharide (LPS)-induced nitric oxide (NO) and tumor necrosis factor (TNF)-α
production in murine macrophage cells (RAW 264.7).6 In addition, ISOLIQ attenuates 6-
hydroxydopamine (6-OHDA)-induced neurotoxicity in mouse embryonic substantia nigra-derived
cell line (SN4741) by regulating mitochondrial function and c-Jun N-terminal kinase (JNK) and
p38 phosphorylation.2 In our previous study, ISOLIQ was isolated from the ethanolic extract of
Glycyrrhiza uralensis and used as a neuroprotectant against 5 mM glutamate-induced oxidative
stress in a mouse-derived hippocampal neuronal cell line (HT22).3
The investigation of drug metabolic fate and metabolites is important in the drug
discovery field, especially for toxicological studies, tracking of the active materials after
biotransformation, and the information for further drug design.7-8 The metabolism of ISOLIQ in
vitro has been evaluated by HPLC-MS analysis in several studies, where the Phase I
metabolites of ISOLIQ were confirmed in human and rat liver microsomes; they are liquiritigenin
(1), 2,4,4,5-tetrahydroxychalcone (2), sulfuretin (3), butein (4), davidigenin (5), cis-6,4-
dihydroxyaurone (6), and trans-6,4-dihydroxyaurone (7) in human and rat liver microsomes.9-10
The major metabolites are 1, 3, 4, and 7, and the minor ones are 2, 5, and 6. The biological
effects of each metabolite have been investigated separately in many studies; they include
osteoblastic cell protection,11 anti-inflammation,12-13 neuroprotection,14 anti-leukemia,15
hepatocytes protection,16 and anti-cancer17 effects. However, there is little information on the
comparison of metabolites’ biological activities in the same batch, especially on hippocampal
3

neuronal cells. Therefore, we synthesized or isolated ISOLIQ Phase I metabolites (Figure 1)
and examined their effect on glutamate-induced HT22 cell death.
OH
HO
OH O
Isoliquiritigenin
HO
OH
OH
OH
OH
HO
HO
O
HO
O
H
O
OH O
O
Sulfuretin (3)
Liquiritigenin (1)
2¢,4,4¢,5¢-Tetrahydroxychalcone (2)
OH
OH
OH
OH
HO
HO
HO
O
H
OH O
OH O
O
Butein (4)
Davidigenin (5)
cis-6,4¢-Dihydroxyaurone (6)
Figure 1. The chemical structures of ISOLIQ and the prepared Phase I metabolites 1 - 6.
4

ISOLIQ was synthesized by aldol condensation as previously described.18 The purified
yellow powder was used for 1H and 13C NMR analysis and the spectral data were compared
with those of in the literature.1
1 was purified from the ethanolic extract of Glycyrrhiza uralensis by silica gel column
chromatography in the previous study and identified its structure without determination of
stereochemistry.19
1
2 was synthesized20 and its NMR spectral data is shown in Table 1. H NMR showed
typical 1,4-disubstituted benzene ring at δ 7.60 and 6.85 (d, J = 8.4 Hz each). Two resonances
with large coupling constants (J = 15.3 Hz) at δ 7.77 and 7.50 suggested that the presence of a
trans-olefinic moiety. In 13C NMR, fifteen carbons including one carbonyl carbon at δ 191.8 were
detected. Four oxygenated aromatic carbons were detected at δ 160.1, 159.9, 154.5, and 150.9.
The peak at δ 144.1 could be assigned to a β-position to the carbonyl group. Combined these
NMR data, 2 was identified as 2,4,4,5-tetarhydroxychalcone.
Table 1
1H and 13C NMR spectral data of synthesized isoliquiritigenin (ISOLIQ) metabolite 2 in CD3OD
(δ in ppm; J Hz)
2
Position
1H
13C
-
112.0
154.5
102.8
159.9
1
2
3
4
-
6.34 s
-
5

-
138.1
5
6
1
7.39 s
114.2
191.8
144.1
117.1
126.5
-
2
7.77 d (15.3)
7.50 d (15.3)
-
3
1
2
3
4
5
6
7.60 d (8.4)
6.85 d (8.4)
-
130.3
115.6
160.1
115.6
130.3
6.85 d (8.4)
7.60 d (8.4)
Chemical shifts (δ; ppm) of 1H NMR (500 MHz) were referenced using tetramethylsilane (TMS) as an internal
standard, and those of 13C NMR (125 MHz) were referenced to the solvent.
3 was synthesized according to the previous report21 with some modifications.22
4 was synthesized by referring to the procedure of ISOLIQ preparation. However, 18.36
mM (2.54 g) 3,4-dihydroxybenzaldehyde was used as a reactant instead of 4-
hydroxybenzaldehyde. Butein (200.00 mg, 0.73 mM) was yielded in the form of an orange
1
powder. The H and 13C NMR spectral data of the product were compared with those in
previous literature.23
5 and 6 were synthesized according to the literature.9
To compare the protective effect of seven compounds against glutamate-induced HT22
cell death, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazlium bromide (MTT) cell viability
6

assay, detection of reactive oxygen species (ROS), and Western blotting experiments were
carried out by using reported methods.19, 24
It is known that a high concentration of glutamate over millimolar can cause oxidative
stress via inhibition of cystine uptake, leading to a lack of glutathione in the cells.25 In this study,
HT22 cells were treated with 5 mM glutamate for 12 h to induce cell death. With this treatment,
the cell viability was reduced to 43.35 ± 1.48% compared to the vehicle control (100.00 ±
2.73%). The cell viability was recovered by 78.93 ± 1.24% in the 10 μM ISOLIQ-pretreated
group. Metabolites 2, 3, and 4 showed higher protective efficacy than ISOLIQ, with treated cells
showing viabilities of 83.51 ± 2.13, 93.73 ± 2.34, and 92.46 ± 1.56%, respectively, at the same
concentration (Figure 2).
7

120
100
80
60
40
0
5
10
15
20
25
Concentration (mM)
Liquiritigenin (1)
2',4,4',5'-Tetrahydroxychalcone (2)
Sulfuretin (3)
Butein (4)
Davidigenin (5)
cis-6,4'-Dihydroxyaurone (6)
ISOLIQ
Figure 2. The comparison of HT22 cell protective effects between ISOLIQ and its metabolites
on 5 mM glutamate-induced cell death. The cell viability was reduced to 43.35 ± 1.48%
compared to the vehicle control (100.00 ± 2.73%). Quercetin (10 μM) was used as a positive
control (93.67 ± 2.50%). The symbols marked as red indicates p< 0.05 compared to the 5 mM
glutamate-only. The value of butein (4) showed statistical difference compared to other
metabolites and ISOLIQ at 5 μM.
8

The intracellular ROS production was significantly increased to 241.63 ± 7.98% after
administration of 5 mM glutamate, whereas the group treated with 5 μM ISOLIQ showed a
decrease to 96.19 ± 2.28% compared to the vehicle control (100.00 ± 8.06%) measured by a
microplate reader. In particular, 5 μM of 2, 3, and 4 significantly decreased the intracellular ROS
production to 137.94 ± 9.50, 102.25 ± 8.57, and 59.79 ± 4.07%, respectively (Figure 3A). In the
flow cytometry analysis, 5 mM of glutamate increased intracellular ROS production, 3.95 ± 0.01-
fold compared to vehicle control (1.00 ± 0.01-fold), while 5 μM ISOLIQ and metabolites 2, 3, and
4 attenuated the glutamate-induced ROS generation to 1.44 ± 0.01, 2.39 ± 0.03, 1.82 ± 0.01,
and 0.69 ± 0.03-fold, respectively (Figure 3B). From these results, it was confirmed that 4 is the
most effective compound among the tested metabolites and the parent compound ISOLIQ at 5
μM.
9

A)
2.5
2.0
1.5
1.0
0.5
0.0
0
5
10
15
20
25
Concentration (mM)
Liquiritigenin (1)
2',4,4',5'-Tetrahydroxychalcone (2)
Sulfuretin (3)
Butein (4)
Davidigenin (5)
cis-6,4'-Dihydroxyaurone (6)
ISOLIQ
10

B)
4
3
2
1
0
0
5
10
15
Concentration (mM)
Figure 3. The inhibitory effects of ISOLIQ and its metabolites on 5 mM glutamate-induced
intracellular ROS production measured by a microplate reader (A) and flow cytometry (B). The
intracellular ROS production was significantly increased to 241.63 ± 7.98% by glutamate
compared to the vehicle control (100.00 ± 8.06%) measured by a microplate reader. In addition,
glutamate increased intracellular ROS production to 3.95 ± 0.01-fold compared to vehicle
control (1.00 ± 0.01-fold) in the flow cytometry analysis. Quercetin (10 μM) was used as a
positive control (53.84 ± 4.98% in Figure 3A; 0.79 ± 0.03-fold in Figure 3B). The symbols
marked as red indicates p< 0.05 compared to the 5 mM glutamate-only. The value of butein (4)
showed statistical difference compared to other metabolites and ISOLIQ at 5 μM.
To investigate the HT22 protective mechanism of the most active compounds, ISOLIQ, 2,
3, and 4, Western blotting was performed. The reduced full-length Bid level by 5 mM glutamate
11

(0.90 ± 0.05-fold; control, 1.00 ± 0.03-fold) was effectively recovered by 2, 3, and 4 at 5 μM.
Especially, the Bid level of 4-treated cells was higher (1.22 ± 0.06-fold) than those of ISOLIQ-
and QC-treated cells (0.94 ± 0.06 and 1.16 ± 0.11-fold, respectively), at 5 μM. The
phosphorylated p38 (pp38) was also elevated by 5 mM glutamate (2.46 ± 0.96-fold) as
compared to the control (1.00 ± 0.38-fold). However, 2, 3, and 4 significantly decreased pp38
level to 1.21 ± 0.50, 1.09 ± 0.52, and 0.75 ± 0.45-fold, respectively. Moreover, the pp38 level of
4-treated cells was lower than that of parent compound ISOLIQ-treated cells (1.03 ± 0.43-fold).
Glutamate also increased the phosphorylated ERK (pERK) level to 3.01 ± 0.30-fold (control,
1.00 ± 0.08-fold), while that was attenuated by 2 (1.64 ± 0.11-fold), 3 (2.02 ± 0.08-fold), and 4
(1.49 ± 0.31-fold), respectively. However, the pERK level was not reduced by three metabolites
as much as ISOLIQ (0.80 ± 0.06-fold) or QC (0.77 ± 0.04-fold). In addition, 2, 3, and 4
significantly suppressed the phosphorylation of JNK induced by 5 mM glutamate (1.84 ± 0.11-
fold; control, 1.00 ± 0.13-fold) to 1.08 ± 0.03, 0.72 ± 0.15, and 0.73 ± 0.36-fold, respectively. The
pJNK levels of 3- or 4-treated cells were lowered than that of ISOLIQ-treated cells (1.00 ± 0.03-
fold; Figure 4). These data demonstrated that 4 had better HT22 protective effects than its
parent compound ISOLIQ, on glutamate-induced cell death resulted by the ROS production, Bid
reduction, and the phosphorylation of p38 and JNK, except for phosphorylation of ERK.
12

A)
+ 5 mM glutamate
1.4
1.2
1.0
0.8
*
*
e
2
3
4
T
C
Q
I
n
N
Q
L
o
C
O
N
S
I
13

B)
+ 5 mM glutamate
4
3
2
1
0
*
*
*
*
*
e
2
3
4
T
C
Q
I
n
N
Q
L
o
C
O
N
S
I
+ 5 mM glutamate
+ 5 mM glutamate
2.5
2.0
1.5
1.0
0.5
0.0
4
3
2
1
0
*
*
*
*
*
*
*
*
*
*
*
*
e
2
3
4
T
C
Q
I
e
2
3
4
T
C
Q
n
N
I
Q
n
L
N
o
Q
L
C
o
C
O
N
O
N
S
I
S
I
Figure 4. The inhibitory effects of ISOLIQ and its three metabolites (2 - 4) on 5 mM glutamate-
induced reduction of full-length Bid (A) and the phosphorylation of mitogen-activated protein
kinases (MAPKs; B). *, p< 0.05 compared to the 5 mM glutamate only.
We demonstrated that butein (4), one of the major Phase I metabolite of ISOLIQ,9-10 has
strong protective activities against 5 mM glutamate-induced cytotoxicity in HT22 cells.
Compared with other compounds, the presence of catechol moiety in B ring was believed to be
the key pharmacophore, as previous study indicated that this moiety is responsible for radical
14

scavenging,26 as well as an inhibitory effect on lipid peroxidation.27 In addition, it has been
discussed that the HT22 cell protection efficacy against glutamate toxicity would be increased
by the presence of two hydroxyl groups attached on C3 and C4 positions (a catechol-bearing B
ring), compared to only one hydroxyl on C4.28 It was also reported that catechol type
compounds exhibited neuroprotective effects via conversion of catechol to a quinone, a kind of
electrophile.29 The electrophiles adduct with the cysteine residues on the Keap1, leading to the
stabilization of nuclear factor erythroid 2-related factor 2 (Nrf2) following its translocation from
cytosol into the nucleus to bind to the antioxidant-responsive element (ARE). The ARE plays a
vital role in the induction of detoxificative Phase II enzymes, such as heme oxygenase-1 (HO-1),
γ-glutamyl cysteine ligase (γ-GCL), and NADPH quinone oxidoreductase 1 (NQO1).30-32 The
cytoprotective mechanisms are subsequently enhanced via regulation of the intracellular redox
status.30-32 There is literature describing that a catechol moiety-bearing diterpene isolated from
Rosmarinus officinalis, carnosic acid, showed the neuroprotective effects against glutamate-
induced toxicity through ARE activation and Phase II enzyme induction mediated by the
transformation of catechol to quinone in the carnosic acid structure.33-34 Metabolite 2, which has
a catechol group on the A ring but not the B ring, also recovered the HT22 cells but the
protective activity was the lowest among three metabolites 2, 3, and 4, indicating that the
position of catechol moiety is more important rather than its presence.
In this study, HT22 cell protective effects of the parent compound ISOLIQ and its
metabolites were compared for the first time. In addition, the metabolite 2 was firstly synthesized
and identified by spectroscopic analysis. Further study on the structure-activity relationship
(SAR) would be helpful for the discovery of ISOLIQ-derived anti-neurodegenerative drug
candidates.
15

Acknowledgements
This work was carried out with the support of “Cooperative Research Program for
Agriculture Science & Technology Development (Project No. PJ011344)” Rural Development
Administration, Republic of Korea.
16

References and notes
1.
Lee, H. K.; Yang, E. J.; Kim, J. Y.; Song, K. S.; Seong, Y. H. Arch. Pharm. Res. 2012,
35, 897.
2.
3.
Hwang, C. K.; Chun, H. S. Biosci. Biotechnol. Biochem. 2012, 76, 536.
Yang, E. J.; Min, J. S.; Ku, H. Y.; Choi, H. S.; Park, M. K.; Kim, M. K.; Song, K. S.; Lee,
D. S. Biochem. Biophys. Res. Commun. 2012, 421, 658.
4.
5.
Maggiolini, M.; Statti, G.; Vivacqua, A.; Gabriele, S.; Rago, V.; Loizzo, M.; Menichini, F.;
Amdo, S. J. Steroid Biochem. Mol. Biol. 2002, 82, 315.
Jung, J. I.; Lim, S. S.; Choi, H. J.; Cho, H. J.; Shin, H. K.; Kim, E. J.; Chung, W. Y.; Park,
K. K.; Park, J. H. J. Nutr. Biochem. 2006, 17, 689.
6.
7.
8.
Lee, S. H.; Kim, J. Y.; Seo, G. S.; Kim, Y. C.; Sohn, D. H. Inflamm. Res. 2009, 58, 257.
Fillet, M.; Frederich, M. Drug Discov. Today Technol. 2015, 13, 19.
Chiu, S. H.; Thompson, K. A.; Vincent, S. H.; Alvaro, R. F.; Huskey, S. W.; Stearns, R.
A.; Pettibone, D. J. Toxicol. Pathol. 1995, 23, 124.
9.
Guo, J.; Liu, D.; Nikolic, D.; Zhu, D.; Pezzuto, J. M.; van Breemen, R. B. Drug Metab.
Dispos. 2008, 36, 461.
10.
Cuendet, M.; Guo, J.; Luo, Y.; Chen, S.; Oteham, C. P.; Moon, R. C.; van Breemen, R.
B.; Marler, L. E.; Pezzuto, J. M. Cancer Prev. Res. (Phila.). 2010, 3, 221.
Suh, K. S.; Rhee, S. Y.; Kim, Y. S.; Choi, E. M. Food Funct. 2014, 5, 1432.
Kim, Y. W.; Zhao, R. J.; Park, S. J.; Lee, J. R.; Cho, I. J.; Yang, C. H.; Kim, S. G.; Kim,
S. C. Br. J. Pharmacol. 2008, 154, 165.
11.
12.
13.
Shin, J. S.; Park, Y. M.; Choi, J. H.; Park, H. J.; Shin, M. C.; Lee, Y. S.; Lee, K. T. Int.
Immunopharmacol. 2010, 10, 943.
14.
15.
Liu, R. T.; Zou, L. B.; Fu, J. Y.; Lu, Q. J. Behav. Brain Res. 2010, 210, 24.
Lee, K. W.; Chung, K. S.; Seo, J. H.; Yim, S. V.; Park, H. J.; Choi, J. H.; Lee, K. T. J.
Cell. Biochem. 2012, 113, 2835.
17

16.
17.
Yang, Y. C.; Lii, C. K.; Lin, A. H.; Yeh, Y. W.; Yao, H. T.; Li, C. C.; Liu, K. L.; Chen, H. W.
Free Radic. Biol. Med. 2011, 51, 2073.
Moon, D. O.; Choi, Y. H.; Moon, S. K.; Kim, W. J.; Kim, G. Y. Toxicol. In Vitro. 2010, 24,
1927.
18.
19.
20.
Deodhar, M.; Black, D. S.; Kumar, N. Tetrahedron. 2007, 63, 5227.
Yang, E. J.; Park, G. H.; Song, K. S. Neurotoxicology. 2013, 39, 114.
Chloromethyl methyl ether (0.14 mL, 1.79 mM) was added to a stirred solution of 2,4,5-
trihydroxyacetophenone (50.00 mg, 0.30 mM) and N,N-diisopropylethylamine (0.31 mL,
1.79 mM) in acetonitrile. The mixture was stirred overnight. In a separate flask, a mixture
of 4-hydroxybenzaldehyde (37.00 mg, 0.30 mM), N,N-diisopropylethylamine (0.11 mg,
0.60 mM), and chloromethyl methyl ether (0.05 mL, 0.60 mM) in acetonitrile was stirred
for 3 h. After the given reaction time, volatiles in the each of the flasks were removed
under reduced pressure, and the residues were each dissolved in ethanol (1.00 mL
each). The ethanolic solutions were mixed together, and a solution of KOH (168.00 mg,
3.00 mM) in water (0.10 mL) was added. The combined mixture was stirred overnight,
and then diluted with ethyl acetate. The mixture was then treated with 1 N-HCl and
extracted with ethyl acetate. Volatiles were removed under the reduced pressure, and
the residue was dissolved in methanol (2.00 mL). 2 N-HCl (0.30 mL) was added and the
reaction mixture was refluxed for 1 h. The resulting mixture was diluted with water and
extracted with ethyl acetate. Volatiles were removed under the reduced pressure, and
the residue was purified with preparative HPLC (YMC-Actus Triart C18, gradient,
acetonitrile/water with 0.1% acetic acid) to yield 2',4,4',5'-tetrahydroxychalcone (2) as a
yellow solid (38.00 mg, 47%); Rf 0.3 (acetone : n-hexane = 1 : 1); IR (ATR-IR) νmax
3333, 3224, 1512, 1160 cm-1; HR-MS calculated for C15H12O5 272.0685, found
272.0683.
18

21.
22.
Shu, C.; Liu, R.; Liu, S.; Li, J. Q.; Yu, Y. F.; He, Q.; Lu, X.; Ye, L. W. Chem. Asian J.
2015, 10, 91.
KOH (2.00 g) in water (2.00 mL) was added to a stirred solution of 6-hydroxy-2,3-
dihydrobenzo[B]furan-3-one (200 mg, 1.33 mM) and 3,4-dihydroxybenzaldehyde (219.00
mg, 1.59 mM) in ethanol (4.00 mL). The mixture was refluxed for 3 h. The resulting
mixture was diluted with water and washed with dichloromethane. 1 N-HCl was added to
the aqueous phase, and the mixture was extracted with ethyl acetate. The organic phase
was dried on anhydrous MgSO4, and volatiles were removed under reduced pressure.
The remaining residue was purified with SiO2 chromatography to yield sulfuretin (3) as
yellow solid (305.00 mg, 85%); Rf 0.2 (ethyl acetate : n-hexane = 1 : 1).
Prachayasittikul, S.; Buraparuangsang, P.; Worachartcheewan, A.; Isarankura-Na-
Ayudhya, C.; Ruchirawat, S.; Prachayasittikul, V. Molecules. 2008, 13, 904.
Kim, S. S.; Seo, J. Y.; Lim, S. S.; Suh, H. J.; Kim, L.; Kim, J. S. Food Sci. Biotechnol.
2015, 24, 333.
23.
24.
25.
Murphy, T. H.; Miyamoto, M.; Sastre, A.; Schnaar, R. L.; Coyle, J. T. Neuron. 1989, 2,
1547.
26.
27.
Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Free Radic. Biol. Med. 1996, 20, 933.
Dugas, A. J., Jr.; Castaneda-Acosta, J.; Bonin, G. C.; Price, K. L.; Fischer, N. H.;
Winston, G. W. J. Nat. Prod. 2000, 63, 327.
28.
29.
30.
31.
32.
Ishige, K.; Schubert, D.; Sagara, Y. Free Radic. Biol. Med. 2001, 30, 433.
Satoh, T.; Lipton, S. A. Trends Neurosci. 2007, 30, 37.
Talalay, P. Biofactors. 2000, 12, 5.
Itoh, K.; Tong, K. I.; Yamamoto, M. Free Radic. Biol. Med. 2004, 36, 1208.
Padmanabhan, B.; Tong, K. I.; Ohta, T.; Nakamura, Y.; Scharlock, M.; Ohtsuji, M.; Kang,
M. I.; Kobayashi, A.; Yokoyama, S.; Yamamoto, M. Mol. Cell. 2006, 21, 689.
19

33.
34.
Satoh, T.; Kosaka, K.; Itoh, K.; Kobayashi, A.; Yamamoto, M.; Shimojo, Y.; Kitajima, C.;
Cui, J.; Kamins, J.; Okamoto, S.; Izumi, M.; Shirasawa, T.; Lipton, S. A. J. Neurochem.
2008, 104, 1116.
Satoh, T.; Izumi, M.; Inukai, Y.; Tsutsumi, Y.; Nakayama, N.; Kosaka, K.; Shimojo, Y.;
Kitajima, C.; Itoh, K.; Yokoi, T.; Shirasawa, T. Neurosci. Lett. 2008, 434, 260.
20

OH
HO
OH O
Isoliquiritigeninꢀ
Phase I metabolitesꢀ
HO
OH
OH
OH
OH
HO
O
O
HO
HO
O
H
OH O
O
Liquiritigenin (1)
Sulfuretin (3)
2ʹ,4,4ʹ,5ʹ-Tetrahydroxychalcone (2)
OH
OH
OH
OH
HO
HO
HO
O
H
OH O
OH O
O
Butein (4)
Davidigenin (5)
cis-6,4ʹ-Dihydroxyaurone (6)
The comparison of HT22 cell protective effects against glutamate toxicityꢀ
Butein (4) is the most active compoundꢀ

Highlights
.
.
.
.
.
Isoliquiritigenin (ISOLIQ) and its six Phase I metabolites were prepared.
The neuroprotective effects of seven compounds were compared on glutamate toxicity.
Butein (4) showed better effects than the parent compound, ISOLIQ.
4 is a chalcone containing a catechol group in the B ring.
The neuroprotective effect of ISOLIQ could be elevated by its metabolite 4.
21