Synthesis and biological evaluation of novel C(6) modified baicalein derivatives as antioxidative agents

Article abstract of DOI:10.1021/jf073224a

Baicalein, one of the major flavones, was found to be responsible for the antioxidative activity of the traditional Chinese medicinal herb Huang-Qin (Scutellaria baicalensis Georgi), which is widely used as an antioxidative, anti-inflammatory, and antitumor agent. The hydroxyl group of the A ring of the baicalein was alkylated at position 6 with terpenoids such as prenyl, geranyl, and farnesyl groups, and their free radical scavenging activities and glutathione (GSH) depletion capacities were examined. Their free radical scavenging activity was measured according to the 2,2′-azinobis(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS^?+) scavenging method. Baicalein and newly synthesized baicalein derivatives were found to be good free radical scavengers. Flow cytometrical method was employed to measure the intracellular antioxidative activity and GSH depletion capacity of these derivatives in human acute monocytic leukemia cell line (THP-1). It was also found that baicalein and its derivatives could decrease the levels of exogenous cumene hydroperoxide and H₂O₂ in THP-1 cells. These compounds also could significantly inhibit the intracellular GSH depletion induced by cumene hydroperoxide in THP-1 cells. The production of cumene hydroperoxide-induced Bax, a pro-apoptotic related protein, could also be inhibited by baicalein and its derivatives. These results suggested that baicalein and its derivatives could be beneficial to human health.

Full text of DOI:10.1021/jf073224a

2838 J. Agric. Food Chem. 2008, 56, 2838–2845
Synthesis and Biological Evaluation of Novel C(6)
Modified Baicalein Derivatives as Antioxidative
Agents
JIN-YI WU,† KING-THOM CHUNG,§ YI-WEN LIU,† FUNG-JOU LU,#
RUEI-SHIUN TSAI,† CHI-HUNG CHEN,‡ AND CHING-HSEIN CHEN*,†
Graduate Institute of Biomedical and Biopharmaceutical Sciences, College of Life Sciences, and Graduate
Institute of Food Science and Biopharmaceutics, National Chiayi University, 300 University Road,
Chiayi 60004, Taiwan; Department of Biology, University of Memphis, Memphis, Tennessee 38152; and
Department of Applied Chemistry, Chung Shan Medical University, Taichung 402, Taiwan
Baicalein, one of the major flavones, was found to be responsible for the antioxidative activity of the
traditional Chinese medicinal herb Huang-Qin (Scutellaria baicalensis Georgi), which is widely used
as an antioxidative, anti-inflammatory, and antitumor agent. The hydroxyl group of the A ring of the
baicalein was alkylated at position 6 with terpenoids such as prenyl, geranyl, and farnesyl groups,
and their free radical scavenging activities and glutathione (GSH) depletion capacities were examined.
Their free radical scavenging activity was measured according to the 2,2′-azinobis(3-ethylbenzothia-
zoline-6-sulfonic acid) (ABTS•+) scavenging method. Baicalein and newly synthesized baicalein
derivatives were found to be good free radical scavengers. Flow cytometrical method was employed
to measure the intracellular antioxidative activity and GSH depletion capacity of these derivatives in
human acute monocytic leukemia cell line (THP-1). It was also found that baicalein and its derivatives
could decrease the levels of exogenous cumene hydroperoxide and H2O2 in THP-1 cells. These
compounds also could significantly inhibit the intracellular GSH depletion induced by cumene
hydroperoxide in THP-1 cells. The production of cumene hydroperoxide-induced Bax, a pro-apoptotic
related protein, could also be inhibited by baicalein and its derivatives. These results suggested that
baicalein and its derivatives could be beneficial to human health.
KEYWORDS: Baicalein; baicalein derivatives; antioxidative; reactive oxygen species; Bax
INTRODUCTION
In the primary cultures of rat hepatocytes, baicalein can quench
the cytotoxicity and genotoxicity of hepatocytes induced by tert-
butyl hydroperoxide (4). Baicalein had also been reported to
repress hydrogen peroxide-induced oxidative stress in human
neuroblastoma HS-SY5Y cells (5). These polyphenols, including
baicalein, may be important in preventing human oxidative
stresses as they have been demonstrated to be capable of
scavenging hydroxyl, 1,1-diphenyl-2-picrylhydrazyl (DPPH),
and alkyl free radicals (6). These flavonoids, including baicalein,
function as scavengers of free radicals by rapid donation of
hydrogen atoms to radicals. Various methods including the 2,2′-
azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) radi-
cal cation scavenging method have been used to estimate the
antioxidative activities of baicalein derivatives in vitro (7, 8).
Polyphenolic compounds, including a large group of fla-
vonoids, are abundant in vegetables, fruits, tea, and Chinese
traditional herbs. Baicalein (5,6,7-trihydroxyflavone), a flavonoid
originating from the roots of Scutellaria baicalensis Georgi
(Huang-Qin), is one of the most important medicinal herbs in
traditional Chinese medicine (1). S. baicalensis Georgi contains
three major polyphenolic components, that is, baicalein, oroxylin
A, and wogonin. Baicalein is known as a selective inhibitor of
12-lipooxygenase, which is responsible for the production of
reactive oxygen species (ROS) during arachidonic acid me-
tabolism (2). Baicalein has been demonstrated to have a strong
activity to scavenge superoxide radicals in cell-free systems (3).
Terpenoid is an antioxidant with a molecular structure
different from that of polyphenols. Several studies demonstrated
the free radical (such as DPPH, superoxide, and lipid peroxide
radicals) scavenging activity of terpenoid (9). Terpenoid could
slow the oxidation of low-density lipid (LDL) (10). Athero-
genesis may be caused by the combining actions of monocyte-
derived macrophages and the oxidized LDL products (11).
Terpenoids were also reported to be responsible for decreasing
* Author to whom correspondence should be addressed (telephone
+886-5-2717837;
fax
+886-5-2717778;
e-mail
chench@
mail.ncyu.edu.tw).
† Graduate Institute of Biomedical and Biopharmaceutical Sciences,
National Chiayi University.
§ University of Memphis.
# Chung Shan Medical University.
‡ Graduate Institute of Food Science and Biopharmaceutics, National
Chiayi University.
10.1021/jf073224a CCC: $40.75  2008 American Chemical Society
Published on Web 03/19/2008

Novel C(6) Modified Baicalein Derivatives
J. Agric. Food Chem., Vol. 56, No. 8, 2008 2839
Scheme 1. Synthesis of Alkoxy Derivatives of Baicalein
ꢀ-amyloid-induced apoptosis in neuronal cells through their
antioxidative properties (12). Some chemicals containing a
terpenoid moiety (group) were reported to have antioxidative
activities (12).
Many human diseases are related to the overproduction of
ROS in living cells. ROS overproduction has been reported to
induce lipid peroxidation, protein and enzyme oxidation, and
DNA damage, which can lead to serious cellular damage such
as mutations, death, and carcinogenesis (13). Moreover, aging,
neurodegeneration, diabetes mellitus, hypertension, and inflam-
matory diseases were also reported to be directly related to ROS
formation inside individual cells (14). Superoxide can be
produced in mitochondrial electron transfer chain or during
phagocytosis and can lead to other cellular reactions to produce
H2O2, hydroxyl radical, and/or lipid peroxide. These ROS can
cause intense cellular damage. Chemical compounds with the
capacity of inhibiting the intracellular oxidative stresses caused
by ROS were often considered to have potential in disease
prevention.
Several studies have demonstrated that ROS generation by
activated monocytes is especially important to exhibit leukocyte-
endothelium interactions in the vascular dysfunction mechanism
(15). Monocytes’ differentiation into macrophages was possibly
triggered by unquenched ROS, which might contribute to
increased inflammatory response such as increased cycloxyge-
nase-2 activity within atheromata (16). It had been reported that
LDL oxidation might play a crucial role in early atherogenesis.
The process of LDL oxidation appears to occur in all major
cells within the arterial wall. Therefore, decreasing the intra-
cellular oxidative stress by baicalein and its derivatives in
monocytes may prevent the formation of monocyte-derived
macrophages; atherogenesis can thus be prevented or minimized.
There are few studies illustrating that baicalein with a
terpenoid moiety is able to increase ROS scavenging activity
and thus decrease intracellular oxidative stresses. In this study,
we synthesized and evaluated a few new baicalein derivatives
that have a substitution at position 6 of the A ring with three
terpenoid groups: prenyl, geranyl, and farnesyl. Our results
indicated that baicalein derivatives with terpenoid groups on
the A ring of the flavones would greatly increase the ROS
scavenging activity and decrease the relative intracellular
oxidative stresses in various ROS-treated human monocyte
THP-1 cells.
NMR. Purity tests of baicalein derivatives were performed by HPLC
equipped with a 280 nm detector and a LiChroCART RP-18e column
(4.6 mm i.d. × 250 mm). The mobile phase was composed of MeOH/
H2O (0.05% TFA) (90:10), and the flow rate was 1.0 mL/min. The
purity of all compounds was >98%.
Bai-C5 (5,6-Dihydroxy-7-prenoxyflavone). Bai-C5 was obtained
as a yellow needle crystal: mp 164-165 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.47 (s, 1H, C5-OH), 8.69 (s, 1H, C7-OH), 8.06 (dd, J
) 8.0, 1.2 Hz, 2H, H2′, H6′), 7.59-7.54 (m, 3H, H3′, H4′, H5′), 6.96
(s, 1H, H3), 6.95 (s, 1H, H8), 5.50 (dd, J ) 8.0, 6.4 Hz, 1H), 4.67 (d,
J ) 6.4 Hz, 2H), 1.77 (s, 3H, CH3), 1.74 (s, 3H, CH3); 13C NMR (100
MHz, DMSO-d6) δ 181.97, 162.84, 153.55, 149.47, 145.89, 137.57,
131.76, 130.66, 130.05, 128.94, 126.13, 119.11, 105.06, 104.54, 92.07,
65.72, 25.54, 18.18.
Bai-C10 (5,6-Dihydroxy-7-geranoxyflavone). Bai-C10 was ob-
tained as a yellow powder: mp 121-124 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.47 (s, 1H, C5-OH), 8.70 (s, 1H, C7-OH), 8.06 (dd, J
) 8.0, 1.2 Hz, 2H, H2′, H6′), 7.59-7.54 (m, 3H, H3′, H4′, H5′), 6.97
(s, 1H, H3), 6.94 (s, 1H, H8), 5.49 (t, J ) 5.6 Hz, 1H), 5.05 (m, 1H),
4.70 (d, J ) 6.4 Hz, 2H), 2.07 (m, 4H), 1.77 (s, 3H, CH3), 1.64 (s, 3H,
CH3), 1.56 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 181.97,
162.84, 153.48, 149.44, 145.88, 140.49, 131.77, 130.86, 130.66, 130.10,
128.94, 126.12, 123.54, 118.95, 105.07, 104.54, 93.74, 92.17, 65.79,
25.78, 25.46, 17.63, 16.51.
Bai-C15 (5,6-Dihydroxy-7-farnesoxyflavone). Bai-C15 was ob-
tained as a yellow powder: mp 103-104 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.47 (s, 1H, C5-OH), 8.71 (s, 1H, C7-OH), 8.05 (dd, J
) 8.0, 1.6 Hz, 2H, H2′, H6′), 7.60-7.54 (m, 3H, H3′, H4′, H5′), 6.96
(s, 1H, H3), 6.92 (s, 1H, H8), 5.48 (t, J ) 6.2 Hz, 1H), 5.05 (t, J ) 6.2
Hz, 1H), 4.96 (t, J ) 6.8 Hz, 1H), 4.71 (d, J ) 6.4 Hz, 2H), 2.06 (m,
4H), 1.93 (m, 2H), 1.85 (m, 2H), 1.75 (s, 3H, CH3), 1.61 (s, 3H, CH3),
1.56 (s, 3H, CH3), 1.49 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6)
δ 182.00, 162.86, 153.46, 149.45, 145.90, 140.52, 134.53, 131.79,
130.68, 130.47, 130.14, 128.97, 126.12, 123.87, 123.32, 119.04, 105.09,
104.54, 92.15, 65.76, 26.20, 25.66, 25.54, 17.59, 16.56, 15.89.
ABTS•+ Decoloration Assay. The antioxidative activity was
measured by the ability of hydrogen-donating antioxidants to scavenge
the ABTS•+ radical cation using a previously reported method (18).
The ABTS•+ radical cation was prepared by mixing a 7 mM ABTS
stock solution with 2.45 mM potassium persulfate (1:1, v/v) and leaving
the mixture for 12–16 h until the reaction was complete and absorbance
was stable. The ABTS•+ solution was diluted in phosphate buffer (PBS)
to an absorbance of 0.70 ( 0.02 at 734 nm for measurement. The
photometric assay was conducted in 1.9 mL of the ABTS•+ solution
and 0.1 mL of baicalein and its derivatives dissolved in an ethanol
solution and mixed for 6 min. Measurements were taken immediately
at 734 nm. The percentage inhibition (expressed as percent decrease
of absorbance at 734 nm) is calculated as a function of concentration
of baicalein derivatives and of Trolox for the standard reference data.
All experiments were carried out in triplicates. The IC50 value was
defined as the concentration that causes a 50% decrease of ABTS•+
radical cation.
MATERIALS AND METHODS
Extraction and Isolation of Baicalein from S. baicalensis Georgi.
Dried S. baicalensis roots were cut into small pieces, immersed, and
extracted with a 10 times volume of acetone twice at room temperature
for 2 weeks. Acetone extracts were concentrated and subjected to
column chromatography on silica gel (7.5 cm i.d. × 30 cm) eluted
with CHCl3 and CHCl3/MeOH (10:1) to yield two fractions. The CHCl3/
MeOH (10:1) eluent was subjected to chromatography on silica gel (3
cm i.d. × 20 cm) and eluted with CH2Cl2/acetone (9:1) to yield baicalein
(2.30 g).
Baicalein (5,6,7-trihydroxyflavone). Baicalein was obtained as a
yellow powder: mp 272-273 °C. [lit. (17) 268-271 °C]; 1H NMR
(400 MHz, DMSO-d6) δ 12.62 (s, 1H, C5-OH), 10.63 (s, 1H, C6-OH),
8.83 (s, 1H, C7-OH), 8.03 (d, J ) 7.2 Hz, 2H, H2′, H6′), 7.61-7.54
(m, 3H, H3′, H4′, H5′), 6.91 (s, 1H, H3), 6.62 (s, 1H, H8).
Chemistry. A series of 6-substituted baicalein derivatives were
synthesized and evaluated by alkylation using alkyl bromide (including
prenyl, geranyl, and farnesyl bromides) with anhydrous potassium
carbonate in anhydrous acetone reflux for 4 h. The synthetic procedure
and reaction conditions for all investigated compounds are shown in
Scheme 1. The synthesized compounds were purified by flash silica
gel column chromatography and the structures based on 1H and 13C
Cell Line and Reagents. The human acute monocytic leukemia cell
line THP-1 was obtained from the American type Culture Collection
(Manassa, VA). Hydroethidine (HE) and chloromethylflourescein
diacetate (CMF-DA) were acquired from Molecular Probes (Eugene,
OR). Propidium iodide (PI), 2′,7′-dichlorodihydrofluorescein diacetate
(DCFH-DA), RPMI-1640 medium, menadione, H2O2, cumene hydro-
peroxide, and other chemicals were purchased from Sigma Chemical
Co. (St. Louis, MO).
Cell Culture and Treatment. The basal medium for the THP-1
cell line was cultured in RPMI-1640 medium supplemented with 10%

2840 J. Agric. Food Chem., Vol. 56, No. 8, 2008
Wu et al.
fetal bovine serum (FBS), 100 units/mL penicillin G, and 100 µg/mL
streptomycin. The stock solution of baicalein and its derivatives was
dissolved in DMSO, and different concentrations (micromolar) were
prepared in the RPMI-1640 basal medium. The final DMSO concentra-
tion was 0.1%.
Analysis of Added Cumene Hydroperoxide Scavenging Activity
of Baicalein and Its Derivatives in THP-1 Cells. THP-1 (106 cells/
mL) cells were incubated with 20 µM DCFH-DA in the 5% CO2
incubator for 10 min, then treated with 5, 10, and 20 µM baicalein and
its derivatives for 10 min at 37 °C in a CO2 incubator, and then treated
with 20 µM cumene hydroperoxide for another 20 min. After treatment
of cumene hydroperoxide, the THP-1 cells were washed with PBS
buffer, collected by centrifugation, and suspended in the same buffer,
and the level of DCF fluorescence was analyzed by flow cytometry.
Analysis of the Intracellular H2O2 Scavenging Activity of
Baicalein and Its Derivatives in THP-1 Cells. THP-1 (106 cells/mL)
cells were incubated with 20 µM DCFH-DA in the 5% CO2 incubator
for 10 min, then treated with 5, 10, and 20 µM baicalein and baicalein’s
derivatives for 10 min at 37 °C in a CO2 incubator, and then treated
with 1600 µM H2O2 for another 30 min. After the H2O2 treatment, the
THP-1 cells were washed with PBS buffer, collected by centrifugation,
and suspended in the same buffer, and the DCF fluorescence was
analyzed by flow cytometry.
Figure 1. Scavenging activities of baicalein and its derivatives on added
cumene hydroperoxide in THP-1 cells. THP-1 cells (106 cells/mL) were
incubated with 20 µM DCFH-DA in the 5% CO2 incubator for 10 min,
then treated with 5, 10, and 20 µM baicalein and baicalein derivatives for
10 min at 37 °C in the 5% CO2 incubator, and then treated with 20 µM
cumene hydroperoxide for another 20 min. Data represent the fluorescence
intensity within the THP-1 cells. Values shown are mean ( standard
deviation (n ) 3–8 of individual experiments). #, statistically significant,
p < 0.05 to untreated group.
Analysis of Intracellular Superoxide Scavenging Activity of
Baicalein and Its Derivatives in THP-1 Cells. THP-1 (106 cells/mL)
cells were incubated with 15 µM HE in the 5% CO2 incubator for 15
min, then treated with 5, 10, and 20 µM baicalein and baicalein’s
derivatives for 15 min at 37 °C in a CO2 incubator, and then treated
with 30 µM menadione for another 30 min. The intracellular NAD(P)H
oxidase could generate superoxide from menadione. After the mena-
dione treatment, the THP-1 cells were washed with PBS buffer,
collected by centrifugation, and suspended in the same buffer, and the
HE fluorescence was analyzed by flow cytometry.
Flow Cytometry Analysis of GSH Content in THP-1 Cells. THP-1
(106 cells/mL) cells were incubated with 10 µM baicalein and
baicalein’s derivatives for 1 h at 37 °C in a CO2 incubator and then
treated with 500 µM cumene hydroperoxide for another 30 min. After
cumene hydroperoxide treatment, the THP-1 cells were incubated with
25 µM CMF-DA for 20 min at 37 °C in a CO2 incubator. After
CMF-DA staining, THP-1 cells were washed with PBS buffer, collected
by centrifugation, and suspended in the PBS buffer, and the CMF
fluorescence was analyzed by flow cytometry.
RESULTS AND DISCUSSION
Intracellular ROS Scavenging Activity of Baicalein and
Its Derivatives. To evaluate the intracellular ROS scavenging
activity of baicalein and its derivatives, three exogenous ROS
sources were applied in the experiments. There were cumene
hydroperoxide, H2O2, and menadione (an intracellular super-
oxide inducer). Two ROS detecting probes, DCFH-DA for
cumene peroxide and H2O2 and HE for superoxide, were used
in this study. As shown in Figure 1, 20 µM cumene hydrop-
eroxide induced about a 2.2-fold increase of the intracellular
DCF fluorescence in THP-1 cells as compared with the untreated
group. Baicalein at 5, 10, and 20 µM concentrations could
decrease the extent of the fluorescence of DCF to about
1.35-1.5-fold of the untreated group. It is of interest to note
that Bai-C5 expressed an appreciable cumene hydroperoxide
scavenging activity; it decreased the DCF fluorescence to less
than that of the untreated group. The cumene hydroperoxide
scavenging activity of Bai-C10 was slightly stronger than that
of baicalein; it exhibited about 1.25-1.30-fold of the untreated
DCF fluorescence. Bai-C15 displayed the weakest cumene
hydroperoxide scavenging activity; the means of DCF fluores-
cence in three concentrations of Bai-C15 were larger than those
of baicalein-treated groups. The order of intracellular cumene
hydroperoxide scavenging activity is Bai-C5 > Bai-C10 >
baicalein > Bai-C15.
Exogenous H2O2 could penetrate into the cellular membrane
to reach the cytosolic or nuclear region and damage directly
the intracellular biomolecules. The intracellular H2O2 scavenging
activities of baicalein derivatives in THP-1 cells were then
evaluated with a DCFH-DA probe. As shown in Figure 2,
baicalein at the three concentrations tested could also depress
the intracellular DCF fluorescence from 2.1- to 1.25-1.35-fold
of the untreated group. The H2O2 scavenging activity of 5 µM
Bai-C5 is similar to that of 5 µM baicalein. Bai-C5 at 10 and
20 µM appeared to have a stronger intracellular H2O2 scavenging
activity than the untreated group, whereas the intracellular DCF
fluorescence were less than that of the untreated group. The
Measurement of Bcl-2, Bax, and Heme Oxygenase-1 (HO-1) by
Western Blotting. THP-1 (106 cells/mL) cells were cultured in 100
mm tissue culture dishes for 24 h. The culture medium was replaced
with new medium and then exposed to 10 µM baicalein and its
derivatives separately for 1 h at 37 °C in the 5% CO2 incubator and
then treated with 500 µM cumene hydroperoxide for another 3 h. After
treatment, cells were washed with PBS, resuspended in protein-extracted
buffer for 10 min, and then centrifuged at 12000g for 10 min at 4 °C
to obtain total extracted proteins (supernatant). The protein concentra-
tions were measured with a commercial kit (Bio-Rad, Richmond, CA).
The Bax, Bcl-2, HO-1, and actin expression was evaluated by Western
blotting analysis. Briefly, the total extracted proteins were boiled in
loading buffer, and an aliquot corresponding to 50 µg of protein was
separated by SDS-PAGE. After blotting, the membranes were incubated
with anti-Bax, anti-Bcl-2, anti-HO-1, and anti-actin antibodies (Labora-
tory Vision, Santa Cruz, CA) overnight and then washed with PBST
solution (0.05% Tween 20 in PBS). Following washing, the second
antibody labeled with horseradish peroxidase was adjacently incubated
for 1 h and then washed with PBST solution (0.05% Tween 20 in PBS).
The antigen–antibody complexes were detected by enhanced chemi-
luminescence (Amersham Pharmacia Biotech, Piscataway, NJ) with a
chemiluminescence analyzer.
Statistical Analysis. Data are presented as means ( standard
deviation from at least three independent experiments and analyzed
using Student’s t test. A P value of <0.05 was considered to be
statistically significant (19).

Novel C(6) Modified Baicalein Derivatives
J. Agric. Food Chem., Vol. 56, No. 8, 2008 2841
Figure 3. Scavenging activities of baicalein and its derivatives on
intracellular superoxide in THP-1 cells. THP-1 (106 cells/mL) cells were
incubated with 15 µM hydroethidine in the 5% CO2 incubator for 15 min,
then treated with 5, 10, and 20 µM baicalein and its derivatives for 15
min at 37 °C in the 5% CO2 incubator, and then treated with 30 µM
menadione for another 30 min. Data represent the fluorescence intensity
within the THP-1 cells. Values shown are mean ( standard deviation
(n ) 3–8 of individual experiments). # and ##, dtatistically significant,
p < 0.05 and p < 0.01 to untreated group, respectively.
Figure 2. Scavenging activities of baicalein and its derivatives on added
H2O2 in THP-1 cells. THP-1 cells (106 cells/mL) were incubated with 20
µM DCFH-DA in the 5% CO2 incubator for 10 min, then treated with 5,
10, and 20 µM baicalein and its derivatives for 10 min at 37 °C in the
5% CO2 incubator, and then treated with 1600 µM H2O2 for another 30
min. Data represent the fluorescence intensity within the THP-1 cells.
Values shown are mean ( standard deviation (n ) 3–8 of individual
experiments). # and ##, statistically significant, p < 0.05 and p < 0.01 to
untreated group, respectively.
H2O2 scavenging activity of Bai-C10 is similar to that of
baicalein; it expressed 1.15-1.20-fold of the untreated DCF
fluorescence. Although Bai-C15 could decrease the H2O2-
induced intracellular DCF fluorescence in THP-1 cells, the H2O2
scavenging activity of Bai-C15 was still weaker than that of
the baicalein. The order of intracellular H2O2 scavenging activity
is Bai-C5 > Bai-C10 > baicalein > Bai-C15.
Superoxide is the first ROS production from mitochondrial
electronic transfer chain. Superoxide overproduction can damage
mitochondrial DNA and other intracellular biomolecules. To
evaluate the intracellular superoxide scavenging activity of
baicalein derivatives, menadione was used as an intracellular
superoxide inducer, and hydroethidine was selected to analyze
the intracellular superoxide in THP-1 cells. As shown in Figure
3, the intracellular superoxide scavenging activity of baicalein
derivatives appeared irregularly. Only 20 µM baicalein and 10
µM Bai-C15 exhibited slight superoxide scavenging activity.
All other concentrations of baicalein and its derivatives did not
inhibit the HE fluorescence to less than that of the untreated
group. These results indicated that the intracellular superoxide
scavenging activity of these derivatives was negligible.
Effect of Baicalein Derivatives in Cumene Hydroperoxide-
Induced Glutathione Depletion. Figures 1-3 indicate that
baicalein derivatives exhibited good cumene hydroperoxide
scavenging activity in THP-1 cells. We further evaluated the
effect of baicalein derivatives on cumene peroxide-induced
glutathione depletion. Glutathione is an important antioxidative
tripeptide; it can cause the intracellular ROS levels to decrease.
Glutathione also plays a detoxifying role to some toxic
substances in cells. Once intracellular glutathione was depleted,
the cells would proceed to die. CMF-DA was used as an
intracellular glutathione probe, and a flow cytometrical method
was employed; the intracellular glutathione depletion can be
expressed in percentage of GSH-negative cells. In Figure 1,
we first screened the intracellular cumene hydroperoxide
scavenging activities among baicalein and its derivatives. The
intracellular DCF fluorescence induced by cumene hydroper-
oxide was set for 2-fold of untreated cells at 1 h of treatment.
Under the experimental condition, the intracellular DCF fluo-
rescence was about 210 in the 20 µM cumene hydroperoxide
treatment as compared with the 100 intracellular DCF fluores-
cence in untreated cells. Originally, we expected that GSH
depletion could be induced by 20 µM cumene hydroperoxide
at 1 h of treatment. Unfortunately, the intracellular GSH
depletion did not markedly increase by 20 µM cumene hydro-
peroxide at 1 h of treatment. These results led us to reexamine
the appropriate concentration and time of cumene hydroperoxide
treatment in intracellular GSH depletion. As shown in Figure
4, 20 µM cumene hydroperoxide did not induce obvious GSH
depletion at 3 h of treatment. The percentage of GSH-negative
cells could be obviously increased to 40.8% in 500 µM cumene
hydroperoxide treatment at 3 h. The 500 µM cumene hydrop-
eroxide and 3 h treatment were used to induce intracellular GSH
depletion. In Figure 5A, 500 µM cumene hydroperoxide
induced 41.1% of GSH-negative cells. In cells treated with
baicalein and its derivatives, the percentages of GSH-negative
cells were <30%. Baicalein and its derivatives could signifi-
cantly decrease the cumene hydroperoxide-induced glutathione
depletion as shown in Figure 5B. There were no significant
differences in the percentages of GSH-negative cells among all
cells treated with either baicalein derivatives or baicalein.
Effect of Baicalein Derivatives on Cumene Hydroperox-
ide-Induced Oxidative Protein Expression. Overproduction
of intracellular ROS could cause various cellular damages
including apoptosis. Bcl-2 and Bax are two important proteins
that regulate the apoptotic process. Bcl-2 is an anti-apoptotic
protein, and its overexpression would prevent apoptosis. Bcl-2
can also neutralize the harmful effects of cellular damage
induced by free radicals, thereby gaining its well-known property
of being an antioxidant (20). Bax is a pro-apoptotic protein (21).
ROS could regulate the Bax expression to trigger apoptosis (22).
We further evaluated Bcl-2 and Bax expressions in cells
pretreated with baicalein and its derivatives after cumene
hydroperoxide treatment. In protein expressions, the 20 µM
cumene hydroperoxide at 1 h of treatment also could not induce
a marked difference as compared with the untreated cells. To

2842 J. Agric. Food Chem., Vol. 56, No. 8, 2008
Wu et al.
Figure 4. Cumene hydroperoxide-induced GSH depletion in THP-1 cells. THP-1 (106 cells/mL) cells were incubated with 0 (untreated), 20, 200, 400,
and 500 µM cumene hydroperoxide for 3 h at 37 °C in the 5% CO2 incubator. After treatment, the THP-1 cells were incubated with 25 µM CMF-DA for
20 min at 37 °C in a CO2 incubator. The CMF fluorescence was analyzed by flow cytometry. Data in each panel represent the percentages of GSH-
negative cells.
Figure 5. Effect of baicalein and its derivatives on cumene hydroperoxide-
induced GSH depletion in THP-1 cells. THP-1 (106 cells/mL) cells were
Figure 6. Cumene hydroperoxide-induced Bax expression in THP-1 cells.
incubated with 10 µM baicalein and baicalein derivatives for 1 h at 37 °C
THP-1 (5 × 106 cells/mL) cells were incubated with 0 (untreated), 100,
in the 5% CO2 incubator and then treated with 500 µM cumene
hydroperoxide for another 3 h. After drug treatment, the THP-1 cells were
300, and 500 µM cumene hydroperoxide for 3 h at 37 °C in the 5% CO2
incubator. After treatment, the THP-1 cells were resuspended in protein-
incubated with 25 µM CMF-DA for 20 min at 37 °C in a CO2 incubator.
(A) Data in each panel represent the percentages of GSH-negative cells.
extracted buffer to obtain total extracted proteins. Fifty micrograms of
protein was separated by SDS-PAGE. The expression of Bax and actin
(B) Data represent the percentages of GSH-negative cells. Values shown
are mean ( standard deviation (n ) 3–8 of individual experiments). #,
was examined by Western blotting. A densitometer was used to quantify
Bax expression. Data are normalized for actin expression.
statistically significant, p < 0.05 to untreated group.
pretreated cells. Three baicalein derivatives caused more severe
induce the obvious protein expression, the concentrations of
cumene hydroperoxide were increased to 100, 300, and 500 µM
and the time of treatment were prolonged to 3 h. As shown in
Figure 6, Bax expression exhibited a concentration-dependent
increase at 3 h of treatment. These results led us to select the
500 µM cumene hydroperoxide and 3 h treatment to evaluate
the related protein expressions. As shown in Figure 7A, both
Bcl-2 and Bax expressions were increased at 3 h after 500 µM
cumene hydroperoxide treatment. In baicalein-treated cells, Bcl-2
expression was equal to that of cumene hydroperoxide-treated
cells. However, Bcl-2 expression was only slightly decreased
in Bai-C5-treated cells but moderately decreased in Bai-C10-
and Bai-C15-treated cells (Figure 7B). In Figure 7C, 500 µM
cumene hydroperoxide also increased Bax expression after 3 h
of treatment. Bax expression was slightly inhibited in baicalein-
inhibitory effects in cumene hydroperoxide-induced Bax expres-
sion as compared with that of baicalein treatment alone. Heme
oxygenase-1 (HO-1) is an oxidative stress defense enzyme,
which could be induced by reactive oxygen species. In Figure
8, 500 µM cumene hydroperoxide could increase HO-1 expres-
sion. HO-1 expression could be slightly decreased by baicalein
and Bai-C5 treatment. In Bai-C10- and Bai-C15-pretreated cells,
HO-1 expression was dramatically decreased after 3 h of cumene
hydroperoxide treatment.
Scavenging Activity of ABTS•+ by Baicalein and Its
Derivatives and Trolox. The ROS scavenging activities of
baicalein and its derivatives were evaluated using a cell-free
system. Generation of the ABTS•+ radical cation forms the basis
of one of the spectrophotomeric methods that have been applied
to the measurement of the antioxidant activity of solution of

Novel C(6) Modified Baicalein Derivatives
J. Agric. Food Chem., Vol. 56, No. 8, 2008 2843
Figure 8. Effect of baicalein and its derivatives on HO-1 expression in
cumene hydroperoxide-treated THP-1 cells. THP-1 (5 × 106 cells/mL)
cells were incubated with 10 µM baicalein and baicalein derivatives for
1 h at 37 °C in the 5% CO2 incubator and then treated with 500 µM
cumene hydroperoxide for another 3 h. After the treatment, the THP-1
cells were resuspended in protein-extracted buffer to obtain total extracted
proteins. Fifty micrograms of protein was separated by SDS-PAGE. (A)
The expression of HO-1 and actin was examined by Western blotting.
(B) A densitometer was used to quantitatively represent the HO-1
expression. Data are normalized for actin expression.
Table 1. Free Radical Scavenging Activity of ABTS•+ by Baicalein, Its
Derivatives, and Trolox
a
IC50 (µM)/ABTS•+
flavone
baicalein
Bai-C5
Bai-C10
Bai-C15
Trolox
5.5 ( 0.40
8.8 ( 0.11
8.7 ( 0.28
10.6 ( 0.50
12.6 ( 0.21
Figure 7. Effect of baicalein and its derivatives on Bcl-2 and Bax
expression in cumene hydroperoxide-treated THP-1 cells. THP-1 (5 ×
106 cells/mL) cells were incubated with 10 µM baicalein and baicalein
derivatives for 1 h at 37 °C in the 5% CO2 incubator and then treated
with 500 µM cumene hydroperoxide for another 3 h. After drug treatment,
the THP-1 cells were resuspended in protein-extracted buffer to obtain
total extracted proteins. Fifty micrograms of protein was separated by
SDS-PAGE. (A) The expression of Bcl-2, Bax, and actin was examined
by Western blotting. A densitometer was used to quantify (B) Bcl-2 and
(C) Bax expression. Data are normalized for actin expression.
a IC50 values were calculated from the concentration at which 50% ABTS•+
free radicals were scavenged by the compounds (n ) 3 or 4) tested. Trolox was
used as a reference antioxidant.
In this cell-free system, the ABTS•+ scavenging activity might
be determined by the number of hydroxyl groups in phenolic
compounds. To view the chemical structure of baicalein, there
are three hydroxyl groups in the phenolic structure. It might let
baicalein express the best ABTS•+ scavenging activity as
compared with its derivatives. In three baicalein derivatives,
there are only two hydroxyl groups present in the phenolic
structure. When one hydroxyl group on C(6) of baicalein was
lost and substituted by a terpenoid group on C(6) of baicalein,
the ABTS•+ scavenging activity was weakened. The results
demonstrated that the order of the ABTS•+ scavenging activity
of baicalein derivatives is Bai-C5 ≈ Bai-C10 > Bai-C15. Of
these baicalein derivatives, Bai-C15 expressed the weakest
ABTS•+ scavenging activity. Bai-C15 contains a large farnesyl
group on C(6) with a lower water solubility as compared with
Bai-C5 and Bai-C10. Because the ABTS•+ is a water-soluble
radical cation, the low water solubility of Bai-C15 might
decrease the ABTS•+ scavenging activity. Only one hydroxyl
group is present in the phenolic structure of Trolox. The ABTS•+
scavenging activity of Trolox was the weakest.
pure substances and aqueous mixtures in vitro (23). The original
ABTS•+ assay was based on the activation of ABTS with
potassium persulfate to form the ABTS•+ radical cation in
aqueous condition. The antioxidant activity reflects the ability
of hydrogen-donating antioxidant to reduce the ABTS•+ radical
cation, compared with that of Trolox, a water-soluble vitamin
E analogue. Among the synthesized 6-position-substituted
baicalein derivatives, the ABTS•+ scavenging activity of ba-
icalein showed a slightly higher ROS scavenging activity than
that of the three baicalein derivative compounds as shown in
Table 1. The IC50 values of ABTS•+ scavenging activities of
baicalein derivatives and baicalein were smaller than that of
Trolox.

2844 J. Agric. Food Chem., Vol. 56, No. 8, 2008
Wu et al.
Table 2. Free Radical Scavenging Activity of ABTS•+ by Prenyl Alcohol,
Geranyl Alcohol, and Farnesyl Alcohol
Baicalein has been demonstrated to decrease the superoxide
production induced by lipopolysaccharide in a rat model (25, 26).
Baicalein has also been demonstrated to have superoxide
scavenging activity in other cell-free experimental systems (27).
When superoxide formation was induced by menadione, only
20 µM baicalein and 10 µM Bai-C15 exhibited some superoxide
scavenging activity. The addition of terpenoid groups on the A
ring of baicalein seemed to be ineffective in increasing the
intracellular scavenging activity of superoxide. Nevertheless,
whether this is true would require further study.
a
IC50 (µM)/ABTS•+
compound
prenyl alcohol (C5H10O)
geranyl alcohol (C10H18O)
farnesyl alcohol (C15H26O)
>200
>200
>200
Glutathione is an antioxidative and detoxifying peptide. A
large number of ROS or toxic substances present in the
intracellular space could induce GSH depletion (28, 29). So far,
there have been few reports on the preventive effects of baicalein
on exogenous ROS-induced intracellular GSH depletion. In our
present study, we demonstrated that baicalein and its derivatives
could significantly decrease the percentage of GSH-negative
cells induced by exogenous cumene hydroperoxide directly.
However, there were no significant differences between baicalein
and its derivatives in preventing GSH depletion after cumene
hydroperoxide treatment (Figure 5B), despite Bai-C5’s exhibit-
ing the best scavenging activity on cumene hydroperoxide
treatment (Figure 1). The differences between GSH depletion
and cumene hydroperoxide scavenging activities of baicalein
and its derivatives might be due to the difference of the
concentrations of cumene hydroperoxide used and the length
of time the experiment was conducted (3 h vs 30 min).
Our present studies demonstrate that 500 µM cumene
hydroperoxide induced Bax and Bcl-2 expression after 3 h of
treatment (Figure 7). Both protein expressions would relate to
apoptosis. ROS are capable of inducing apoptosis through Bax
overexpression. To prevent ROS damage, the cells usually
would increase Bcl-2 or HO-1 expressions as a defense system.
It is worth noting that three baicalein derivatives were better
than baicalein in inhibiting Bax expression. The Bcl-2 and HO-1
expression levels of the cells treated with three baicalein
derivatives were also lower than that of baicalein-treated cells.
In Bai-C15-pretreated cells, Bax expression was even lower than
that in the untreated cells. This result contradicted the result in
cumene hydroperoxide scavenging activity (Figure 1). One
possible explanation is that the farnesyl group in Bai-C15 is
more hydrophobic than the prenyl group in Bai-C5 and the
geranyl group in Bai-C10. Due to the large size of the Bai-C15
molecule, cell membrane penetration might require a longer
time, such as 3 or 4 h of treatment, to manifest its effect.
Conclusion. In conclusion, the presence of prenyl, geranyl,
or farnesyl groups at position 6 of the A ring of baicalein
derivatives would affect their antioxidative capabilities, which
might be beneficial to human health. The in-depth mechanisms
of these effects will require further study.
a IC50 values were calculated from the concentration at which 50% ABTS•+
free radicals were scavenged by the compounds (n ) 3 or 4) tested.
To clarify the difference of scavenging activities among
prenyl, geranyl, and farnesyl bromides, their radical scavenging
activities were evaluated by ABTS•+ decoloration assay. Prenyl,
geranyl, and farnesyl bromide can form prenyl, geranyl, and
farnesyl alcohol in aqueous condition, because bromide is a good
leaving group in organic synthesis. Therefore, we measured the
radical scavenging activity of prenyl, geranyl, and farnesyl
alcohol to prove that there were no effects of scavenging activity
due to the length of carbohydrate of the terpenoid moiety. As
shown in Table 2, the IC50 of prenyl, geranyl, and farnesyl
alcohol on ABTS•+ scavenging activity was larger than 200
µM. There were no differences of scavenging activities between
prenyl, geranyl, and farnesyl alcohol. These results explain the
difference of intracellular scavenging activities against cumene
hydroperoxide or H2O2 among baicalein, Bai-C5, Bai-C10, and
Bai-C15 as perhaps being due to the lengths of carbohydrate
of the terpenoid. The length of carbohydrate of the terpenoid
in Bai-C10 and Bai-C15 is longer than that of Bai-C5 and may
delay the penetration rates of Bai-C10 and Bai-C15 into the
intracellular space.
The A ring on baicalein is often chemically modified to
produce new drugs. For example, alkylation of baicalein on the
A ring could increase the activity of anti-P-glycoprotein, a
multiple drug resistant gene product (24). The ROS scavenging
activity and effects of oxidative stress-related protein expression
of baicalein and its derivatives in cellular systems had not been
well addressed despite baicalein’s being a well-known antioxi-
dant. We showed for the first time the antioxidative effects of
baicalein and its terpenoid derivatives on the A ring in human
THP-1 cells.
In the cellular system, Bai-C5 was most effective in scaveng-
ing intracellular cumene hydroperoxide and H2O2 among the
compounds tested. The cell membrane is a hydrophobic
environment because it contains a large number of phospholipid
and protein molecules. The terpenoid groups are hydrophobic.
The addition of terpenoid groups on the A ring of baicalein
derivatives might increase the hydrophobicity of the molecule
and prompt these baicalein derivatives to penetrate readily
through the cell membrane into the intracellular space. Our
results demonstrated that Bai-C5 exhibited the best scavenging
activity in THP-1 cells after cumene hydroperoxide or H2O2
treatment. However, the cumene hydroperoxide or H2O2 scav-
enging activity of Bai-C10 or Bai-C15 treatment was weaker
than that of Bai-C5 treatment. This might be due to the fact
that the length of carbohydrate of the terpenoid in Bai-C10 and
Bai-C15 is longer than that of Bai-C5; it may hinder their rates
of penetration into the intracellular space. This would explain
why Bai-C15 expressed the weakest scavenging activity for
cumene hydroperoxide or H2O2 among the three baicalein
derivatives within a short period of 30 min of treatment.
ABBREVIATIONS USED
ROS, reactive oxygen species; GSH, glutathione; DCF, 2′,7′-
dichlorofluorescein; HE, hydroethidine; CMF, chloromethylfluo-
rescein; DMEM, Dulbecco’s modified Eagle’s medium; FBS,
fetal bovine serum; HO-1, heme oxygenase-1.
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