Synthesis, anti-inflammatory, and antioxidant activities of 18β-glycyrrhetinic acid derivatives as chemical mediators and xanthine oxidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.02.025

Twenty 18β-glycyrrhetic acid (18β-GA) derivatives 2-21 including 13 new 18β-GA derivatives were synthesized and evaluated as anti-inflammatory and antioxidant agents. Compounds 7 and 20 with a 3,4-seco-structure and compound 6 with a lactone moiety showed potent inhibitory effect on superoxide anion generation in rat neutrophils response to fMLP/CB and PMA, respectively. Compound 6 with a lactone moiety revealed stronger inhibitory effect on XO activity than those of compounds 13 and 14 with a 3,4-seco-struture. Compound 14, a 30-isoproylcarbamoyl seco-compound exhibited potent inhibitory effect on NO accumulation and iNOS protein expression while compounds 3, 10, 13, 15, 17, and 21 revealed potent inhibitory effect on tumor necrosis factor-α (TNF-α) formation in RAW 264.7 cells in response to lipopolysaccharide (LPS). The cleavage of ring A of 3 attenuated the inhibitory effect on TNF-α formation in RAW 264.7 cells in response to LPS except for 17. The present results suggested these compounds were potential to be served as anti-inflammatory and antioxidant agents.

Full text of DOI:10.1016/j.bmc.2009.02.025

Bioorganic & Medicinal Chemistry 17 (2009) 2785–2792
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, anti-inflammatory, and antioxidant activities of 18b-glycyrrhetinic
acid derivatives as chemical mediators and xanthine oxidase inhibitors
Dravidum Maitraie a, , Chi-Feng Hung b, , Huang-Yao Tu a, Ya-Ting Liou a, Bai-Luh Wei c,
,
*
Shyh-Chyun Yang a,e, Jih-Pyang Wang d, Chun-Nan Lin a,c,e,
*
a Faculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan
b School of Medicine, Fu Jen Catholic University, Taipei Hsien 242, Taiwan
c Institute of Life Science, National Taitung University, Taitung 950, Taiwan
d Department of Education and Research, Taichung Veterans General Hospital, Taichung 407, Taiwan
e Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty 18b-glycyrrhetic acid (18b-GA) derivatives 2–21 including 13 new 18b-GA derivatives were syn-
thesized and evaluated as anti-inflammatory and antioxidant agents. Compounds 7 and 20 with a 3,4-
seco-structure and compound 6 with a lactone moiety showed potent inhibitory effect on superoxide
anion generation in rat neutrophils response to fMLP/CB and PMA, respectively. Compound 6 with a lac-
tone moiety revealed stronger inhibitory effect on XO activity than those of compounds 13 and 14 with a
3,4-seco-struture. Compound 14, a 30-isoproylcarbamoyl seco-compound exhibited potent inhibitory
effect on NO accumulation and iNOS protein expression while compounds 3, 10, 13, 15, 17, and 21
Received 26 December 2008
Revised 15 February 2009
Accepted 18 February 2009
Available online 21 February 2009
Keywords:
18b-Glycyrrhetinic acid derivatives
Synthesis
Cytotoxicity
Anti-inflammatory activity
Antioxidant activity
revealed potent inhibitory effect on tumor necrosis factor-
response to lipopolysaccharide (LPS). The cleavage of ring A of 3 attenuated the inhibitory effect on
TNF- formation in RAW 264.7 cells in response to LPS except for 17. The present results suggested these
compounds were potential to be served as anti-inflammatory and antioxidant agents.
Ó 2009 Elsevier Ltd. All rights reserved.
a (TNF-a) formation in RAW 264.7 cells in
a
1. Introduction
The oxidative damage of DNA in the development of certain can-
cers and lipid oxidative damage in the occurrence and progression
of vascular disease are also associated with ROS.6
XO is a key enzyme that catalyzes the oxidation of xanthine and
hypoxanthine into uric acid and plays a vital role in causing hyper-
uricemia and gaut.8
Mast cells and neutrophils, stimulated with various inducers
may contribute to inflammatory disorders. Macrophages are
important in nonspecific host resistant to microbial pathogens
and serve as central regulators of the specific immune response.1
Following the activation of macrophage, TNF-
a
and NO were gen-
Synthetic oleanane and ursane with various enone functional-
erated in response to LPS.2,3 NO plays a central role in macrophage-
induced cytotoxicity and excess NO may contribute to the patho-
ities in ring A were reported to have anti-inflammatory effect.9
A
several of natural triterpenoids, such as 18b-GA, an oleanane triter-
penoid, showed remarkably active against the edema produced by
12-O-tetradecanoylphorbol acetate (TPA).10 Natural oleanolic acid
and ursolic acid possessed nonenzymatic antioxidative and anti-
glycative properties.11 Here, we report the preparation of a series
of 18b-GA derivatives from 18b-GA (1). The structure modification
was focused on cleavage of ring A and introduction of various ester
groups at C-3 and C-30 for biological evaluation. The structure and
biological activity of 18b-GA derivatives relationship also reported
in the present paper.
physiology of septic shock.4 TNF-
a is an important pro-inflamma-
tory cytokine with immune and inflammatory functions and
generally considered as principal mediator of septic shock. The
overexpression of TNF-
a is associated with autoimmune diseases
such as rheumatoid arthritis and Crohn’s disease.5
Reactive oxygen species (ROS) is associated in pathological
events including inflammation, metabolic disorders, cellular aging,
reperfusion damages, artherosclerosis, and carcinogenesis.6 ROS
induce programmed cell death or necrosis, induce or suppress
the expression of many genes, and activate cell signaling cascades.7
2. Results and discussion
2.1. Chemistry
* Corresponding authors. Tel.: +886 089 318855; fax: +886 089 351554 (B.-L.W.);
tel.: +886 7 3121101; fax: +886 7 5562365 (C.-N.L.).
E-mail addresses: blwei@cc.nttu.edu.tw (B.-L. Wei), lincna@cc.kmu.edu.tw (C.-N.
Lin).
Compounds 2–21 were synthesized as depicted in Schemes 1–3.
Starting material, 18b-GA (1) was oxidized to 3-keto compound (2)
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.025

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D. Maitraie et al. / Bioorg. Med. Chem. 17 (2009) 2785–2792
Scheme 1. Reagents and conditions: (a) CrO3, DMF, rt, 12 h; (b) CH3OH, H2SO4, reflux, 48 h; (c) m-CPBA, CH2Cl2, rt, 12 h; (d) appropriate alcohol, p-TSA, rt, 6–8 h; (e) EDCI, 4-
dimethylaminopyridine (DMAP), CH2Cl2, MeOH, rt, 6–8 h.
COOR
CONHR'
COOH
H
H
H
O
O
O
f
f
H3COOC
H3COOC
H3COOC
R'-NH
2
ROH
12 R = CH(CH3)2
13 R = CH2-Ph
8
14 R' = CH(CH3)2
15 R' = C6H5
Scheme 2. Reagents and conditions: (f) EDC, DMAP, CH2Cl2, ROH or R0-NH2, rt, 24 h.
using CrO3 in DMF.12 The treatment of 3-oxo-derivative 2 with m-
chloroperbenzoic acid (m-CPBA) afforded lactone 5.13 Methylation
of 18b-GA with excess of MeOH in the presence of H2SO4 as cata-
lyst to yield 3. The oxidation of C-3–OH of 3 to give 4. Compound
4 was further treated with m-CPBA to give 30-methyl ester lactone
(6). This compound was synthesized in a different synthetic route,
by the esterification of lactone (5) used 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide (EDCI) as the activating agent in metha-
nol.14 The lactone ring of 5 or 6 was cleaved by treatment of p-
toluenesulfonic acid (p-TSA) in appropriate solvent to yield prod-
ucts 7–11. Treatment of seco-methyl ester 8 with isopropyl alcohol
and benzyl alcohol in the presence of EDCI and 4-dimethylamino-
pyridine (DMAP) provided the corresponding 30-ester compounds
12 and 13, respectively. Similar treatment of 8 with amines, isopro-
pylamine, and aniline afforded 30-amide seco-compounds 14 and
15, respectively (Scheme 2). Compounds 16, 18, and 19 were pre-
pared from lactone (5) which was the common starting material
for synthesis of these compounds. Acid group at C-30 was modified
to ester and amide, respectively, followed by ring cleavage in the
presence of p-TSA in methylene chloride yielded C-30 substituted
seco-acids 17, 20, and 21 (Scheme 3).
2.2. Biological results and discussion
The degranulation of mast cells and neutrophils, and macro-
phages contribute to inflammatory disorder. Combining potent
inhibition of chemical mediators released from mast cells, neutro-
phils, and macrophages would suggest a promising anti-inflamma-
tory agents.18 The anti-inflammatory effects of 1–20 were studied
in vitro for their inhibitory effects on chemical mediators released
from mast cells, neutrophils, and macrophages. Synthetic com-
pounds 1–20 did not show significant inhibitory effects on mast
cell and neutrophil degranulation.
FMLP (0.3 lM)/CB (5 lg/mL) or PMA (3 nM) stimulated super-
oxide anion generation in rat neutrophils. As shown in Figures 1
and 2, positive control (diphenylene iodonium chloride, DPI), 2,
7, and 20 and DPI, 6, 13, and 14 had potent and concentration-
dependent inhibitory effects on fMLP/CB and PMA induced super-
oxide anion generation with IC50 values of 2.7 2.0, 10.3 5.2,
7.0 2.0, and 9.8 5.2 and 1.8 0.4, 12.9 1.8, 17.0 1.5, and
15.6 1.7 lM (Table 2), respectively while the other compounds
had no significant inhibitory effects (data not shown). These results
indicate that reduction of the carbonyl group at C-3 of 2 to 1 or
introduction of a lipophilic alkyl group at C-30 of 18b-GA deriva-
tives might attenuate their inhibitory effects on fMLP/CB-induced
responses while introduction of a lipophilic group at C-30 of 5 or
a benzylester moiety at C-30 such as 13 or an isopropylcarbamoyl
group at C-30 such as 14 significantly enhance the inhibitory ef-
The known 18b-GA derivatives 2–4, 5, 7, 8, 11, and 22 were
identified by spectroscopic data and compared with those of data
reported in literatures.14–17 The new 18b-GA derivatives were
characterized by various spectroscopic methods (Table 1 and Sec-
tion 4) and compared with data reported in literatures.14–17

D. Maitraie et al. / Bioorg. Med. Chem. 17 (2009) 2785–2792
2787
COOCH2Ph
COOCH2Ph
H
H
O
O
g
HOOC
f
COOH
O
ROH
O
H
O
16
17
CONHR'
CONHR'
O
H
H
O
O
O
f
g
HOOC
5
R'-NH
O
2
O
18 R' = CH(CH3)2
19 R' = C6H5
20 R' = CH(CH3)2
21 R' = C6H5
Scheme 3. Reagents and conditions: (f) EDC, DMAP, CH2Cl2, ROH or R0-NH2, rt, 24 h; (g) CH2Cl2, p-TSA, rt, 24 h.
fects on PMA-induced responses. As shown in Figure 1 also reveals
that cleavage of ring A of 2 to afford compounds with 3,4-seco-
structure, such as 7, enhances its inhibitory effect on fMLP/CB-in-
duced responses. Introduction of a lipophilic moiety at C-30 or
C-3 such as 14, 15, or 21 weakened the inhibitory activity on
fMLP/CB-induced response but introduction of an isopropylcarba-
moyl moiety at C-30 such as 20 enhances the inhibitory activity
on response induced by same inducer (Fig. 1). As shown in Figure 2,
lactone derivative from 18b-GA such as 6 displays stronger and
concentration-dependent effect on inhibitory effect on PMA-in-
duced response than those of 13 and 14. The result indicated that
cleavage of ring A of 6 and introduction of a side chain greater than
methyl group at C-30 attenuate the inhibitory effect on PMA-in-
duced response. Because fMLP and PMA activate NADPH oxidase
to produce superoxide anion through different cellular signaling
mechanism.19 These 18b-GA derivatives indicated different inhibi-
tory effect on fMLP/CB- and PMA-induced responses. These results
supported that above observations.
trol (genistein), 3, 10, 13, 15, 17, and 21 strongly and concentration
dependently inhibited TNF- generation in LPS-stimulated RAW
264.7 cells with IC50 values of 26.5 9.1, 1.3 0.4, 26.1 14.7,
13.7 4.3, 15.5 3.2, 2.3 0.5, 27.7 7.2 M (Table 2), respec-
tively. Among them the suppression of TNF- generation in RAW
a
l
a
264.7 cells stimulated with LPS by these compounds except for
21 were stronger than that of positive control. The esterification
of C-30 at 18b-GA enhances the suppressed effect on TNF-
ation while reduction of C-3–OH of 18b-GA or cleavage of ring A
attenuates the inhibition of TNF- generation on LPS-stimulated
a gener-
a
response. The esterification of C-30 at 3,4-seco-type derivatives
from 18b-GA with benzyl alcohol strongly enhances the inhibitory
effect on TNF-a generation while amidation with isopropylamine
attenuate the inhibition of TNF-a generation on LPS-stimulated
response.
ROS have been known to damage many biological macromole-
cules, with DNA being a significant target. The ability of 18b-GA
ꢀÅ
derivatives to inhibit the DNA damage caused by O2 [generated
Treatment of RAW 264.7 macrophage-like cells with LPS (1
l
g/
by xanthine (XA)/XO] was studied in vitro by agarose gel electro-
phoresis.22 As shown in Figure 6, compounds 2, 6, 7, 13, 14, and
20 revealed significant inhibitory effects on superoxide anion for-
mation released from rat neutrophils activated by fMLP/CB or
PMA and showed significant protective effects on oxidative DNA
damage caused by O2ꢀÅ. The above results clearly revealed that
these compounds may possess 1,1-diphenyl-2-picrylhydrazl
(DPPH), a stable free radical that accepts an electron or hydrogen
radical to become a stable diamagnetic molecules,22 scavenging
activity or inhibitory effect on XO, an enzyme that catalyzed the
oxidation of XA and hypoxanthine into uric acid,8 activity. For
determined the antioxidant activities of these compounds, the rad-
ical scavenging activities and XO inhibitory activities of these com-
pounds were analyzed. As shown in Figure 7, compounds 6, 13, 14,
22, and allopurinol (positive control) significantly inhibit the XO
activity in a concentration-dependent manner with IC50 values of
mL) for 24 h induced NO production as assessed by measuring the
accumulation of nitrite, a stable metabolite of NO, in the media
based on Griess reaction.20 As shown in Figure 3, LPS induced a sig-
nificant increase of NO production and this effect was concentra-
tion dependently suppressed by positive control (N-(3-
aminometyl)benzylacetamidine, 1400 W) and 14 with IC50 values
of 1.5 0.2 and 13.1 5.0
pounds 2, 11, 12, and 13 each at 30
l
M (Table 2), respectively while com-
M indicated about 40% (Table
l
2) of inhibitory effect on NO accumulation in the media induced by
LPS. The above observation shows that a lipophilic group substi-
tuted at C-3 or C-30 of 3,4-seco-type derivatives from 18b-GA
might enhance the suppression of NO accumulations in the media
induced by LPS except for compound 15. To determine whether the
inhibition of NO production in RAW 264.7 cells is attributed to the
decrease of iNOS protein expression, Western blotting analysis was
performed. Unstimulated cells expressed very low level of iNOS
protein, whereas LPS 50 ng/mL induced a large amount of iNOS
protein expression (Fig. 4). Compound 14 significantly inhibited
the iNOS protein expression. Thus, the blockade of iNOS transcrip-
tion has a critical role as evidenced from the parallelism of the
inhibition of NO production and iNOS protein expression by 14.
131.5 2.7, 175.0 0.9, 192.4 2.7, 186.1 1.0, and 2.0 0.7 lM,
respectively. Compounds 2, 7, and 20 weakly inhibit the XO activ-
ity (data not shown). All the compounds used for study on inhibi-
tory effects on XO activity did not show DPPH scavenging activity.
The XO inhibitors 6, 13, and 14 also significantly suppressed the
superoxide anion formation in rat neutrophils stimulated with
PMA. This result showed the suppression of superoxide anion for-
mation by these compound in rat neutrophils stimulated with PMA
Effect on the generation of TNF-
a was determined in RAW 264.7
cells stimulated with LPS.2,3,21 As shown in Figure 5, positive con-

2788
D. Maitraie et al. / Bioorg. Med. Chem. 17 (2009) 2785–2792
Table 1
13C NMR spectral data for compounds 6, 9, 10, and 12–21
Position
6
9
10
23.7
12
23.7
13
23.7
14
23.7
15
23.8
16
38.6
17
23.8
18
38.7
19
38.6
20
23.7
21
23.5
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
38.7
32.1
23.5
31.8
31.0
179.6
146.4
38.6
29.0
34.1
40.3
50.9
38.5
199.6
128.3
169.5
45.0
26.3
26.4
31.6
48.3
43.6
43.9
31.1
37.7
114.2
23.3
31.4
175.7
146.5
38.7
28.6
34.4
43.6
52.8
41.1
199.6
128.3
169.7
45.0
26.3
26.5
31.7
48.3
43.7
50.8
31.0
37.6
114.1
23.4
31.3
176.1
146.4
38.6
28.4
34.3
43.9
52.6
41.1
199.4
128.2
169.2
44.9
26.3
26.4
31.6
48.0
43.5
50.7
31.0
37.5
114.1
23.4
31.4
174.6a
146.5
38.7
28.6
34.4
43.3
52.9
42.1
199.6
128.3
169.6
45.0
26.4
26.5
31.8
48.1
41.1
43.7
31.4
37.4
114.2
23.4
31.4
173.9a
146.5
38.8
28.4
34.4
43.7
52.8
42.0
199.5
128.5
169.3
45.1
26.4
26.5
32.0
48.1
44.5
50.8
31.6
37.4
114.2
23.4
32.0
176.1
85.6
54.5
22.0
32.4
43.4
61.2
39.6
198.8
128.6
169.2
45.3
26.3
26.4
31.8
48.1
41.0
43.9
31.1
37.6
32.2
25.9
31.4
179.0
146.5
38.7
28.4
34.2
43.9
52.9
41.1
199.5
128.3
169.4
45.0
26.4
26.5
31.7
48.1
43.6
51.1
31.2
37.6
114.3
23.4
32.1
175.5
85.6
54.2
22.0
32.2
43.3
61.3
39.4
198.7
128.3
169.6
45.2
26.2
26.3
31.4
48.0
41.7
43.2
31.8
37.3
31.8
25.9
S15
32.0
175.6
85.6
54.5
22.0
32.4
43.4
61.3
39.6
198.7
128.7
169.1
45.3
26.4
26.4
31.7
48.0
41.8
44.5
32.2
37.4
32.2
25.9
31.4
179.0
146.4
38.7
28.6
34.1
43.3
52.8
41.9
199.8
128.3
170.0
45.1
26.4
26.5
31.8
48.2
41.1
43.7
31.4
37.4
114.3
23.3
31.4
180.6
146.3
39.1
28.8
34.1
44.0
52.5
41.7
200.6
128.5
170.7
45.3
26.4
26.7
31.8
48.4
44.6
50.4
31.6
37.5
114.2
23.2
175.4
85.5
54.2
22.0
32.0
43.3
61.2
39.4
198.7
128.4
169.3
45.2
26.2
26.2
31.7
48.2
41.0
43.8
30.9
37.6
31.7
25.9
173.5
146.6
38.7
29.7
34.3
43.6
52.7
40.9
199.8
128.5
169.4
45.1
26.4
26.5
31.4
48.2
40.9
43.8
30.8
37.7
114.2
23.4
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
OCH3
17.4
18.0
23.0
28.2
28.1
176.7
51.6
19.5
18.6
23.8
28.5
28.4
181.8
19.4
18.6
23.4
28.5
28.2
176.9
51.7
19.5
18.6
23.3
29.2
28.2
19.4
18.5
23.2
29.1
28.1
174.2
51.4
19.5
18.6
23.2
19.5
18.6
23.3
17.4
18.2
23.1
28.4
28.2
175.6
19.4
18.6
23.3
28.9
28.3
176.2
17.4
18.0
23.0
29.3
28.5
174.5
17.4
18.2
23.1
29.3
28.4
173.9
19.5
18.6
23.2
29.4
29.1
174.8
19.4
18.5
23.1
29.7
29.1
174.5
29.5
29.4
29.2
29.2
174.2
51.5
174.3a
51.5
174.3a
51.5
OCH(CH3)2
50.6
67.3
OCH(CH3)2
21.8, 21.8
21.6, 21.8
OCH2
66.1
136.0
128.3
128.5
128.3
128.5
128.3
66.2
136.0
128.2
128.5
128.3
128.5
128.2
66.2
136.1
128.2
128.6
128.4
128.6
128.2
10
137.8
120.1
129.0
124.4
129.0
120.1
137.8
120.2
128.7
124.4
128.7
120.2
137.9
121.4
128.7
124.4
128.7
121.4
20
30
40
50
60
NH–CH(CH3)2
NH–CH(CH3)2
50.8
22.7
22.9
41.0
22.6
22.7
50.8
22.6
22.9
a
Signals may be interchanged in the same column.
100
2
7
20
80
DPI
60
40
20
0
0.1
1
10
100
Concentration (µM)
Figure 1. The inhibitory effects of 2, 7, 20, and DPI on superoxide anion generation
in rat neutrophils stimulated with fMLP/CB. Data are presented as means SD
(n = 3–6). Diphenylene iodonium chloride (DPI) was used as positive control.
Figure 2. The inhibitory effects of 6, 13, 14, and DPI on superoxide anion generation
in rat neutrophils stimulated with PMA. Data are presented as means SD (n = 3–6).
DPI was used as positive control.

D. Maitraie et al. / Bioorg. Med. Chem. 17 (2009) 2785–2792
2789
Table 2
100
The inhibitory effect of 18b-GA derivatives on the superoxide anion formation from
rat neutrophils stimulated with fMLP/CB or PMA (A), the accumulation of NO2 in
3
ꢀ
10
13
15
17
21
genistein
RAW 264.7 cells stimulated with LPS (B), and TNF-
stimulated with LPS (C)
a
formation from RAW 264.7 cells
80
60
40
20
0
a
a
a
Compound
IC50
fMLP/CB
10.3 5.2
(l
M) (A)
IC50
(
lM) (B)
IC50
(lM) (C)
PMA
RAW 264.7
RAW 264.7
2
>30 (45.6 9.1)
3
1.3 0.4
6
12.9 1.8
7
7.0 2.0
10
26.1 14.7
13.7 4.3
11
12
13
14
>30 (44.3 10.5)
>30 (43.0 4.3)
>30 (44.5 4.2)
13.1 5.0
17.0 1.5
15.6 1.7
15
17
15.5 3.2
2.3 5.0
0.1
1
10
100
20
21
DPI
1400 W
Genistein
9.8 5.2
2.7 2.0
Concentration (µM)
27.7 7.2
1.8 0.4
Figure 5. The inhibitory effects of 3, 10, 13, 15, 17, 21, and genistein on the
formation of TNF- in the culture media of RAW 264.7 cells in response to LPS. Data
1.5 0.2
a
26.5 9.1
are presented as means SD (n = 3–6). Genistein was used as positive control.
Diphenylene iodonium chloride (DPI), N-(3-aminomethyl)benzylacetamidine (1400
W), and genistein were used as positive controls for A, B, and C, respectively.
a
When 50% inhibition could not be reached at the highest concentration, the % of
inhibition is given in parentheses. Data are presented as means SD (n = 3–6).
100
2
11
80
12
13
14
ꢀÅ
Figure 6. Inhibition of DNA strand breaks induced by O2 (generated by XA/XO) in
60
1400W
the presence of 2, 6, 7, 13, 14, and 20 studied by gel electrophoresis. Supercoiled
plasmid pBR322 DNA (500 ng) in phosphate buffer (pH 7.4) solution was incubated
for 20 min with XA/XO acting as the control. Lane 1, DNA (without XA/XO); lane 2,
40
20
0
control; lane 3, control + SOD (300
serving as positive control; lane 5, control + 2 (300
(300 M); lane 7, control + 7 (300 M); lane 8, control + 13 (300
control + 14 (300 M); lane 10, control + 20 (300 M).
l
M); lane 4, control + quercetin (300
M); lane 6, control + 6
M); lane 9,
lM)
l
l
l
l
l
l
phils stimulated with fMLP/CB did not correlate with XA/XO
system. The above results also indicated that the cleavage of lac-
tone ring of 6 attenuates the inhibitory effect on XO activity.
-20
0.01
0.1
1
10
100
Concentration (µM)
3. Conclusion
Figure 3. The inhibitory effects of 2, 11–14, and 1400 W on the accumulation of
NO2 in the culture media of RAW 264.7 cells in response to LPS. Data are presented
ꢀ
Twenty 18b-GA derivatives were synthesized and biologically
evaluated as agents with antioxidant and anti-inflammatory activ-
ities. Compounds 2, 3, 6, 7, 10, 13, 14, 15, 17, 20, 21, and 22 exert
potent inhibitory effects on the release of chemical mediators from
inflammatory cells and compounds 6, 13, 14, and 22 display anti-
oxidant and anti-inflammatory activities.
as means SD (n = 3–6). N-(3-Aminomethyl)benzylacetamidine) (1400 W) was
used as positive control.
NO plays a central role in macrophage induced cytotoxicity and
has been demonstrated to implicate in the pathology of central
neurologic disease and in the peripheral tissue damage associated
with acute and chronic inflammation18 and septic shock.4 The
present study suggests that the inhibition of NO generation and
suppression of superoxide anion formation by 14 in RAW 264.7
cells and rat neutrophils, respectively, and also the inhibition of
XO activity and protective effect on oxidative DNA damage by 14
may have value in the therapeutical treatment or prevention of
certain central as well as peripheral inflammatory diseases associ-
Figure 4. Effect of 14 on the expression of iNOS protein. RAW 264.7 cells were
pretreated with 14 at 3 lM or 10 lM for 1 h, followed by stimulation with 50 ng/mL
ated with the increase of NO production. The suppression of TNF-
a
of LPS. After 24 h, the expression of iNOS protein was analyzed by Western blotting.
Similar results were obtained from three independent experiments.
production by compounds 3, 10, 13, 17, and 21 in macrophage may
also have value in the treatment or prevention of certain inflam-
matory diseases or septic shock associated with increase of TNF-
may correlate with XA/XO system while the suppression of super-
oxide anion formation by compounds 2, 7, and 20 in rat neutro-
a production.

2790
D. Maitraie et al. / Bioorg. Med. Chem. 17 (2009) 2785–2792
5.72 (1H, s, H-12). 13C NMR (CDCl3): see Table 1. EIMS (70 eV) m/
z (% rel. int.): 526 [M]+ (2). HRESIMS: Calcd for C33H50O5Na:
549.3556. Found: 549.3554.
4.2.3. 3,4-seco-11-Oxo-18b-olean-4(23),12-dien-3,30-dioic acid
30-methyl ester (10)
Compound 6 (0.1 g, 0.2 mmol) in CH2Cl2 (10 mL) was added
0.3 g of p-toluenesulfonic acid. The mixture was stirred at room
temperature for 6–8 h. The mixture was diluted with water and ex-
tracted with CHCl3. The organic layer was washed with 5% sodium
bicarbonate solution, dried over MgSO4, filtered, and concentrated.
The residue was purified by a column to give 10 as white solid
(0.08 g, 0.16 mmol, 78%): mp 89–94 °C; ½a D25
ꢁ
171 (c 0.1, CHCl3). IR
(KBr): 1729, 1658 cmꢀ1 1H NMR (CDCl3): d 0.78 (3H, s, Me-28),
.
1.12 (3H, s, Me-29), 1.13 (6H, s, Me-25 and Me-26), 1.35 (3H, s,
Me-27), 1.72 (3H, s, Me-24), 3.66 (3H, s, –COOCH3), 4.66 (1H, br
s, H-23), 4.86 (1H, br s, H-23), 5.65 (1H, s, H-12). 13C NMR (CDCl3):
see Table 1. EIMS (70 eV) m/z (% rel. int.): 498 [M]+ (10). HRESIMS:
Calcd for C31H46O5Na: 521.3243. Found: 521.3245.
Figure 7. Dose-dependent inhibition of XO by 6, 13, 14, and allopurinol. Data are
presented as means s.e.m., n = 6.
4.2.4. General procedure for esterification and amidation of
lactone and seco-compounds
4. Experimental
4.1. Chemistry
To a solution of lactone or seco-compound (1 mmol) in dry
CH2Cl2 (10 mL) were added EDCI (2 mmol) and DMAP (catalytic
amount) followed by alcohol or amine (2 mmol). The reaction mix-
ture was stirred at room temperature overnight. After the reaction
was finished (monitored by TLC) the mixture was diluted with
water and extracted with chloroform. The organic solution was
washed with 3% hydrochloric acid solution, brine, dried over
MgSO4, and filtered. The solvent was removed in vacuo and puri-
fied by column chromatography.
Melting points (uncorrected) were determined with a Yanco
Micro-Melting point apparatus. IR spectra were determined with
a
Perkin–Elmer system 2000 FTIR spectrophotometer. 1H
(400 MHz) and 13C (100 MHz) NMR were recorded on a Varian
UNITY-400 spectrometer. Low-resolution mass spectra and high-
resolution mass spectra data were obtained on a JMX-HX 100 mass
spectrometer. Chromatography was performed using a flash-col-
umn technique on Silica Gel 60 supplied by E. Merck.
4.2.5. 3,4-seco-11-Oxo-18b-olean-4(23),12-dien-3,30-dioic acid
3-methyl,30-isopropyl ester (12)
Compound 12 was prepared from 8 following the general proce-
dure described for esterification. Compound 12 was obtained as
4.2. Synthesis of 18b-glycyrrhetinic acid derivatives
light yellow solid (0.28 g, 0.52 mmol, 52%): mp 116–119 °C; ½a D25
ꢁ
4.2.1. 4-Hydroxy-3,4-seco-11-oxo-18b-olean-12-en-3,30-dioic
acid 3,4 lactone 30 methyl ester (6)
126 (c 0.1, CHCl3). IR (KBr): 1724, 1658 cmꢀ1 1H NMR (CDCl3): d
.
0.79 (3H, s, Me-28), 1.11 (3H, s, Me-29), 1.14 (3H, s, Me-25), 1.15
(3H, s, Me-26), 1.21 (3H, d, J = 6.4 Hz, –CHCH3), 1.24 (3H, d,
J = 6.4 Hz, –CHCH3), 1.37 (3H, s, Me-27), 1.74 (3H, s, Me-24), 3.60
(3H, s, OCH3), 4.67 (1H, br s, H-23), 4.87 (1H, br s, H-23), 5.02
(1H, septet, J = 6.4 Hz, OCH), 5.63 (1H, s, H-12). 13C NMR (CDCl3):
see Table 1. EIMS (70 eV) m/z (% rel. int.): 540 [M]+ (18). HRESIMS:
Calcd for C34H52O5Na: 563.3712. Found: 563.3711.
Compound 4 (1 g, 2.1 mmol) in CH2Cl2 (30 mL) was added 3-
chloroperoxybenzoic acid (3.6 g, 21.3 mmol). The mixture was al-
lowed to stand at room temperature in dark for 12 h. The solution
was diluted with CHCl3, washed with 5% KI solution and 5% sodium
sulfite solution, dried over Na2SO4, and concentrated to give 6, as
white solid (0.82 g, 1.6 mmol, 78%): mp 166–171 °C; ½a D25
ꢁ
189 (c
0.1, CHCl3). IR (KBr): 1715, 1648 cmꢀ1 1H NMR (CDCl3): d 0.67
.
(3H, s, Me-28), 1.10 (3H, s, Me-29), 1.11 (3H, s, Me-26), 1.33 (6H,
s, Me-25 and Me-27), 1.40 (3H, s, Me-23), 1.43 (3H, s, Me-24),
3.64 (3H, s, –OCH3), 5.65 (1H, s, H-12). 13C NMR (CDCl3): see Table
1. EIMS (70 eV) m/z (% rel. int.): 498 [M]+ (3). HRESIMS: Calcd for
C31H46O5Na: 521.3243. Found: 521.3241.
4.2.6. 3,4-seco-11-Oxo-18b-olean-4(23),12-dien-3,30-dioic acid
3-methyl,30-benzyl ester (13)
Compound 13 was prepared from 8 following the general proce-
dure described for esterification using benzyl alcohol as alcohol
moiety. Compound 13 was obtained as light yellow solid (0.35 g,
0.6 mmol, 60%): mp 32–38 °C; ½a D25
ꢁ
120 (c 0.1, CHCl3). IR (KBr):
4.2.2. 3,4-seco-11-Oxo-18b-olean-4(23),12-dien-3,30-dioic acid
3-isopropyl ester (9)
1730, 1657 cmꢀ1 1H NMR (CDCl3): d 0.72 (3H, s, Me-28), 1.12
.
(3H, s, Me-29), 1.14 (3H, s, Me-25), 1.15 (3H, s, Me-26), 1.35 (3H,
s, Me-27), 1.74 (3H, s, Me-24), 3.59 (3H, s, –OCH3), 4.67 (1H, br s,
H-23), 4.87 (1H, br s, H-23), 5.07 (1H, d, J = 12 Hz, –CHH–C6H5),
5.19 (1H, d, J = 12 Hz, CHH–C6H5), 5.55 (1H, s, H-12), 7.35 (5H, m,
C6H5). 13C NMR (CDCl3): see Table 1. EIMS (70 eV) m/z (% rel.
int.): 588 [M]+ (3). HRESIMS: Calcd for C38H52O5Na: 611.3712.
Found: 611.3709.
Compound 5 (0.1 g, 0.2 mmol) in isopropyl alcohol (5 mL),
CH2Cl2 (2 mL) was added 0.3 g of p-toluenesulfonic acid. The mix-
ture was stirred at room temperature for 6–8 h, concentrated in va-
cuo, poured into water, extracted with CHCl3. The CHCl3 layer was
washed with 5% sodium bicarbonate and brine, dried over Na2SO4,
and concentrated. The residue was purified by a column to yield 9,
as light yellow solid (0.06 g, 0.12 mmol, 60%): mp 94–99 °C; ½a D25
ꢁ
154 (c 0.1, CHCl3). IR (KBr): 2976, 1727, 1658, 1459, 1382,
4.2.7. Methyl 30-isopropylcarbamoyl-11-oxo-18b-3,4-seco-
olean-4(23),12-dien-3-oate (14)
1175 cmꢀ1 1H NMR (CDCl3): d 0.85 (3H, s, Me-28), 1.16 (3H, s,
.
Me-29), 1.17 (3H, s, Me-25), 1.19 (3H, s, Me-26), 1.21 (6H, d,
J = 6.4 Hz, CH(CH3)2), 1.39 (3H, s, Me-27), 1.76 (3H, s, Me-24),
4.69 (1H, br s, H-23), 4.89 (1H, br s, H-23), 4.95 (1H, m, –OCH),
Compound 14 was prepared from 8 following the general proce-
dure described for amidation using iso-propyl amine as amine moi-
ety. Compound 14 was obtained as light yellow solid (0.34 g,

D. Maitraie et al. / Bioorg. Med. Chem. 17 (2009) 2785–2792
2791
0.63 mmol, 63%): mp 80–85 °C; ½a D25
ꢁ
116 (c 0.1, CHCl3). IR (KBr):
4.2.12. 4-Hydroxy-3,4-seco-11-oxo-18b-olean-12-en-3,30-dioic
3,4-lactone 30-phenylcarbamate (19)
3379, 1720, 1645 cmꢀ1
.
1H NMR (CDCl3): d 0.82 (3H, s, Me-28),
1.11 (3H, s, Me-29), 1.13, 1.15 (6H, each d, J = 6.4 Hz, –CH(CH3)2),
1.15 (3H, s, Me-26), 1.16 (3H, s, Me-25), 1.39 (3H, s, Me-27), 1.75
(3H, s, Me-24), 3.62 (3H, s, OCH3), 4.11 (1H, m, NCH), 4.68 (1H,
br s, H-23), 4.89 (1H, br s, H-23), 5.37 (1H, d, J = 8.4, –NH), 5.64
(1H, s, H-12). 13C NMR (CDCl3): see Table 1. EIMS (70 eV) m/z (%
rel. int.): 539 [M]+ (34). HRESIMS: Calcd for C34H53NO4Na:
562.3872. Found: 562.3874.
Compound 19 was synthesized from 5 following the general
procedure described for amidation using aniline as one of the reac-
tant. Compound 19 was obtained as white solid (0.37 g, 0.66 mmol,
66%): mp 123–128 °C; ½a D25
ꢁ
268 (c 0.1, CHCl3). IR (KBr): 3359, 1717,
1655 cmꢀ1. 1H NMR (CDCl3): d 0.81 (3H, s, Me-28), 1.16 (3H, s, Me-
29), 1.26 (3H, s, Me-26), 1.37 (3H, s, Me-27), 1.41 (3H, s, Me-25),
1.44 (3H, s, Me-23), 1.48 (3H, s, Me-24), 5.75 (1H, s, H-12), 7.11
(1H, m, aromatic proton), 7.34 (3H, m, NH, and aromatic proton),
7.50 (2H, m, aromatic proton). 13C NMR (CDCl3): see Table 1. EIMS
(70 eV) m/z (% rel. int.): 559 [M]+ (20). HRESIMS: Calcd for
C36H49NO4Na: 582.3559. Found: 582.3557.
4.2.8. Methyl 30-phenylcarbamoyl-11-oxo-18b-olean-3,4-seco-
olean-4(23),12-dien-3-oate (15)
Compound 15 was prepared from 8 with the general procedure
described for amidation using aniline as amine moiety. Compound
15 was obtained as light yellow solid (0.38 g, 0.66 mmol, 66%): mp
4.2.13. 30-Isopropylcarbamoyl-11-oxo-18b-3,4-seco-olean-
4(23),12-dien-3-oic acid (20)
149–154 °C; ½a 2D5
ꢁ
192 (c 0.1, CHCl3). IR (KBr): 3375, 1728, 1655,
1598 cmꢀ1
.
1H NMR (CDCl3): d 0.83 (3H, s, Me-28), 1.15 (3H, s,
Compound 20 was prepared from 18 following the procedure
described for synthesis of 10. Compound 20 was obtained as light
Me-25), 1.16 (3H, s, Me-26), 1.26 (3H, s, Me-29), 1.41 (3H, s, Me-
27), 1.75 (3H, s, Me-24), 3.63 (3H, s, OCH3), 4.69 (1H, br s, H-23),
4.89 (1H, br s, H-23), 5.72 (1H, s, H-12), 7.11 (1H, brt, J = 8.4 Hz,
aromatic H), 7.33 (2H, br t, J = 8.4 Hz, aromatic H), 7.37 (1H, br s,
NH), 7.51 (1H, dd, J = 8.4 Hz, 1.2 Hz, aromatic H). 13C NMR (CDCl3):
see Table 1. EIMS (70 eV) m/z (% rel. int.): 573 [M]+ (32). HRESIMS:
Calcd for C37H51NO4Na: 596.3716. Found: 596.3718.
yellow solid (0.06 g, 0.12 mmol, 62%): mp 142–146 °C; ½a D25
ꢁ
126 (c
0.1, CHCl3). IR (KBr): 3348, 1738, 1651 cmꢀ1 1H NMR (CDCl3): d
.
0.81 (3H, s, Me-28), 1.11 (6H, d, J = 6.8 Hz, –CH(CH3)2), 1.12 (3H,
s, Me-29), 1.15 (6H, s, Me-25, and Me-26), 1.37 (3H, s, Me-27),
1.74 (3H, s, Me-24), 4.11 (1H, m, –CH(CH3)2), 4.68 (1H, br s, H-
23), 4.88 (1H, br s, H-23), 5.64 (1H, s, H-12), 5.71 (1H, d,
J = 8.0 Hz, NH). 13C NMR (CDCl3): see Table 1. EIMS (70 eV) m/z (%
rel. int.): 525 [M]+ (32). HRESIMS: Calcd for C33H51NO4Na:
548.3716. Found: 548.3718.
4.2.9. 4-Hydroxy-3,4-seco-11-oxo-18b-olean-12-en-3,30-dioic
3,4-lactone 30-benzylester (16)
Compound 16 was synthesized from 5 following the general
procedure described for esterification using benzyl alcohol as one
of the reactant. Compound 16 was obtained as white solid
4.2.14. 30-Phenylcabamoyl-11-oxo-18b-3,4-seco-olean-
4(23),12-dien-3-oic acid (21)
(0.36 g, 0.62 mmol, 62%): mp 83–89 °C; ½a D25
ꢁ
254 (c 0.1, CHCl3). IR
Compound 21 was prepared from 19 following the procedure
described for synthesis of 10. Compound 21 was obtained as white
(KBr): 1726, 1654 cmꢀ1 1H NMR (CDCl3): d 0.73 (3H, s, Me-28),
.
1.14 (3H, s, Me-29), 1.16 (3H, s, Me-26), 1.36 (3H, s, Me-25), 1.37
(3H, s, Me-27), 1.44 (3H, s, Me-23), 1.47 (3H, s, Me-24), 5.08 (1H,
d, J = 12.4 Hz, –OCHH–), 5.21 (1H, d, J = 12.4 Hz, –OCHH–), 5.58
(1H, s, H-12), 7.36 (5H, m, aromatic proton). 13C NMR (CDCl3):
see Table 1. EIMS (70 eV) m/z (% rel. int.): [M]+ 574 (15). HRESIMS:
Calcd for C37H50O5Na: 597.3556. Found: 597.3553.
solid (0.07 g, 0.13 mmol, 65%): mp 162–166 °C; ½a D25
ꢁ
262 (c 0.1,
CHCl3). IR (KBr): 3365, 1652, 1526 cmꢀ1 1H NMR (CDCl3): d 0.84
.
(3H, s, Me-28), 0.97 (3H, s, Me-25), 1.00 (3H, s, Me-26), 1.31 (3H,
s, Me-29), 1.40 (3H, s, Me-27), 1.69 (3H, s, Me-24), 4.62 (1H, br s,
H-23), 4.86 (1H, br s, H-23), 5.84 (1H, s, H-12), 7.07 (1H, m, aro-
matic proton), 7.28 (2H, m, aromatic proton), 7.45 (2H, m, aromatic
proton), 8.27 (1H, br s, NH). 13C NMR (CDCl3): see Table 1. EIMS
(70 eV) m/z (% rel. int.): 559 [M]+ (64). HRESIMS: Calcd for
C36H49NO4Na: 582.3559. Found: 582.3558.
4.2.10. 3,4-seco-11-Oxo-18b-olean-4(23),12-dien-3,30-dioic
acid 30-benzylester (17)
Compound 17 was prepared from 16 following the procedure
described for synthesis of 10. Compound 17 was obtained as white
4.3. Mast cell degranulation, neutrophil degranulation,
superoxide anion generation, macrophage culture and drug
treatment, NO determination, and Western blotting analysis
solid (0.08 g, 0.14 mmol, 70%): mp 87–92 °C; ½a D25
ꢁ
145 (c 0.1,
CHCl3). IR (KBr): 1726, 1657 cmꢀ1 1H NMR (CDCl3): d 0.73 (3H, s,
.
Me-28), 1.15 (3H, s, Me-29), 1.16 (6H, s, Me-25 and 26), 1.36
(3H, s, Me-27), 1.75 (3H, s, Me-24), 4.69 (1H, br s, H-23), 4.89
(1H, br s, H-23), 5.09 (1H, d, J = 12.4 Hz, –OCHH–), 5.21 (1H, d,
J = 12.4 Hz, –OCHH–), 5.57 (1H, s, H-12), 7.37 (5H, m, C6H5). 13C
NMR (CDCl3): see Table 1. EIMS (70 eV) m/z (% rel. int.): [M]+ 574
(25). HRESIMS: Calcd for C37H50O5Na: 597.3556. Found:
597.3558.
Compound stock solution (30 mM in DMSO) was prepared and
stored at ꢀ25 °C, and was diluted with DMSO to 1–20 mM range at
room temperature before experiment. The final percentage of
DMSO in the reaction mixture was less than 0.5% (v/v). Rat (Spra-
gue Dawley) peritoneal mast cells23 and peripheral blood neutro-
phils24 were isolated and incubated with test compounds for
5 min at 37 °C before stimulation with 10
lg/mL of compound
4.2.11. 4-Hydroxy-3,4-seco-11-oxo-18b-olean-12-en-3,30-dioic
3,4-lactone 30-isopropylcarbamate (18)
48/80 for another 15 min or with 1 M formyl-Met-Leu-Phe (fMLP)
l
for another 45 min, respectively. The degranulation of mast cells
and neutrophils was assessed by the determination of histamine
and b-glucuronidase, and b-glucuronidase and lysozyme, respec-
tively, in the supernatant.23,25 The total content of lysozyme and
b-glucuronidase was measured from the Triton X-100-treated
cells. In the superoxide anion generation experiments, neutrophils
Compound 18 was synthesized from 5 following the general
procedure described for amidation using iso-propylamine as one
of the reactant. Compound 18 was obtained as light yellow solid
(0.32 g, 0.6 mmol, 60%): mp 94–99 °C; ½a D25
ꢁ
154 (c 0.1, CHCl3). IR
(KBr): 3382, 1720, 1653 cmꢀ1 1H NMR (CDCl3): d 0.77 (3H, s,
.
Me-28), 1.07 (3H, s, Me-29), 1.11 (3H, s, Me-26), 1.33 (3H, s, Me-
27), 1.34 (3H, s, Me-25), 1.40 (3H, s, Me-23), 1.44 (3H, s, Me-24),
4.07 (1H, m, CH(CH3)2), 5.52 (1H, d, J = 8.0 Hz, NH), 5.64 (1H, s,
H-12). 13C NMR (CDCl3): see Table 1. EIMS (70 eV) m/z (% rel.
int.): 525 [M]+ (27). HRESIMS: Calcd for C33H51NO4Na: 548.3716.
Found: 548.3717.
were stimulated with fMLP (0.3 lM)/CB (5 lg/mL) for 30 min in
the presence of cytochrome c, and the superoxide anion generation
was measured in terms of superoxide dismutase-inhibitable cyto-
chrome c reduction.26,27 Murine macrophage-like cell line RAW
264.7 cells were plated in 96-well plate, and incubated with test
compounds for 1 h at 37 °C before stimulation with 1 lg/mL of

2792
D. Maitraie et al. / Bioorg. Med. Chem. 17 (2009) 2785–2792
lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) in the cell med-
ium was determined by the Griess reaction.28 In Western blot anal-
ysis, cells were washed with PBS twice and harvested in Laemmli
sodium dodecyl sulfate (SDS) sample buffer. Cell lysates were sep-
arated by 10% SDS–PAGE, and electrophoretically transferred to
poly(vinylidene difluoride) membranes. Membranes were blocked
for 1 h at room temperature in TBST buffer (10 mM Tris–HCl, pH
8.0, 150 mM NaCl and 0.1% Tween 20) containing 5% nonfat milk.
Membranes were washed with TBST buffer and then incubated
for 1 h with a monoclonal anti-iNOS antibody (1:1000 dilution).
Following washed with TBST buffer, horseradish peroxidase-la-
beled anti-mouse IgG (1:10,000 dilution) was added at room tem-
perature for 1 h. The blots were developed using ECL Western
4.6. Statistical analysis
Data were expressed as means SD. Statistical analysis were
performed using the Bonferroni t-test method after ANOVA for
multigroup comparison and the student’s t-test method for two
group comparison, with p < 0.05 was considered to be statistically
significant.
Acknowledgment
This work was supported by a research grant from the National
Science Council of the Republic of China (NSC95-2320-B-037-037)
and partial supported by a fund of KMU-EM-97-2.1.b.
blotting reagents.22 Secretory levels of TNF-
a in culture superna-
tants were determined by the EIA kit according to the procedure
of manusfacturer.
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4.4. Inhibition of oxidative DNA damage
A mixture of supercoiled plasmid pBR322 DNA (1
xanthine (2 mM)/xanthine oxidase (0.7 U/mL) in 10 mM phos-
phate buffer (pH 7.4) was incubated for 20 min with 500 M of
superoxide dismutase, quercetin, or 2, 6, 7, 13, 14, and 20 in a to-
tal volume of 20 L in a 1.5 mL microfuge tube at 37 °C, respec-
tively. Quercetin was used as positive control. After incubating
for 20 min, a 15 L aliquot of mixture was loaded into 1.0% aga-
rose gel containing ethidium bromide (0.05 g/mL) in Tris-ace-
tate-ethylenediaminetetraacetic acid (EDTA) buffer. The
lg/lL) and
l
l
l
l
electrophoresis was carried out for 30 min at 100 V. Then the gels
were illuminated with UV light and photographed. Plasmid DNA
subjected to electrophoresis without superoxide dismutase, quer-
cetin, or 2, 6, 7, 13, 14, and 20 served as the control. The gel elec-
trophoretic motility of the various forms of DNA was compared
with the control.6
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4.5. Assay of xanthine oxidase activity
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The xanthine oxidase activity with xanthine as the substrate
was measured at 25 °C, according to the protocol of Kong and asso-
ciates29 with modification. The assay mixture consisting of 50
lL of
test solution, 60
lL of 70 mM phosphate buffer (pH 7.5), and 30
lL
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of enzyme solution [0.1 U/mL in 70 mM phosphate buffer (pH 7.5)]
was prepared immediately before use. After preincubation at 25 °C
for 15 min, the reaction was initiated by addition of 60
lL of sub-
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500.
strate solution (150 M xanthine in the same buffer). The reaction
was monitored for 5 min at 295 nm. The xanthine oxidase activity
was expressed as micromoles of uric acid per minute.
l