Phloroglucinols Inhibit Chemical Mediators and Xanthine Oxidase, and Protect Cisplatin-Induced Cell Death by Reducing Reactive Oxygen Species in Normal Human Urothelial and Bladder Cancer Cells

Article abstract of DOI:10.1021/jf900935n

Phloroglucinols, garcinielliptones HA-HE (1-5), and C (6) were studied in vitro for their inhibitory effects on chemical mediators released from mast cells, neutrophils, and macrophages. Compound 6 revealed significant inhibitory effect on release of lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB). Compounds 3, 4, and 6 showed significant inhibitory effects on superoxide anion generation in rat neutrophils stimulated with (fMLP)/(CB), while compounds 1 and 5 revealed inhibitory effects on tumor necrosis factor-α (TNF-α) formation in macrophages stimulated with lipopolysaccharide (LPS). Compounds 1 and 3-6 showed inhibitory effects on xanthine oxidase (XO) and could inhibit the DNA breakage caused by O₂^-.. Treatment of NTUB1 with 2 to 60 μM compound 3 and 5 μM cisplatin and SV-HUC1 with 9 to 60 μM 3 and 5 μM cisplatin, respectively, resulted in an increase of viability of cells. These results indicated that compounds 1 and 3-6 showed anti-inflammatory effects and antioxidant activities. Compound 3 mediates through the suppression of XO activity and reduction of reactive oxygen species (ROS), and protection of subsequent cell death.

Full text of DOI:10.1021/jf900935n

8782 J. Agric. Food Chem. 2009, 57, 8782–8787
DOI:10.1021/jf900935n
Phloroglucinols Inhibit Chemical Mediators and Xanthine
Oxidase, and Protect Cisplatin-Induced Cell Death by Reducing
Reactive Oxygen Species in Normal Human Urothelial and
Bladder Cancer Cells
†
K
AI-WEI
L
H
IN
,
†,3 A-MEI
OUR,‡ BAI-LUH
EONG-SHIAU
H
UANG
,
‡,3 HUANG-YAO
T
U
,† JING-R
U
W
ENG
,
T
ZYH-CHYUAN
W
EI,§ SHYH-CHYUN
Y
ANG,*,† JIH-PYANG
,†,§,#
WANG
,
^
Y
P
U,
AND
C
HUN-NAN
L *
IN
†School of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ‡Institute of Biochemistry,
College of Medicine, Kaohsiung Medical University 807, Taiwan, §Department of Life Science, National
Taitung University, Taitung 950, Taiwan, Department of Education and Research, Taichung Veterans
General Hospital, Taichung 407, Taiwan, ^Department of Urology, College of Medicine, National Taiwan
University, Taipei 100, Taiwan, and #Center of Excellence for Environmental Medicine, Kaohsiung
Medical University, Kaohsiung 807, Taiwan. 3These authors contributed equally to this work.
Phloroglucinols, garcinielliptones HA-HE (1-5), and C (6) were studied in vitro for their inhibitory
effects on chemical mediators released from mast cells, neutrophils, and macrophages. Compound
6 revealed significant inhibitory effect on release of lysozyme from rat neutrophils stimulated with
formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB). Compounds 3, 4, and 6 showed significant
inhibitory effects on superoxide anion generation in rat neutrophils stimulated with (fMLP)/(CB),
while compounds 1 and 5 revealed inhibitory effects on tumor necrosis factor-R (TNF-R) formation in
macrophages stimulated with lipopolysaccharide (LPS). Compounds 1 and 3-6 showed inhibitory
effects on xanthine oxidase (XO) and could inhibit the DNA breakage caused by O-2 •. Treatment of
NTUB1 with 2 to 60 μM compound 3 and 5 μM cisplatin and SV-HUC1 with 9 to 60 μM 3 and 5 μM
cisplatin, respectively, resulted in an increase of viability of cells. These results indicated that
compounds 1 and 3-6 showed anti-inflammatory effects and antioxidant activities. Compound 3
mediates through the suppression of XO activity and reduction of reactive oxygen species (ROS),
and protection of subsequent cell death.
KEYWORDS: Phloroglucinol; garcinielliptones HA-HE and anti-inflammatory; antioxidant; NTUB1;
SV-HUC1
INTRODUCTION
macrophage-induced cytotoxicity, and excess of NO may induce
pathophysiological septic shock (9). TNF-R is an important pro-
inflammatory cytokine with immune and inflammatory functions
and is generally considered as a principal mediator of septic
shock. The overexpression of TNF-R is associated with auto-
immune diseases such as rheumatoid arthritis (10).
XO is a key enzyme that catalyzes the oxidation of xanthine
and hypoxanthine into uric acid and displays a vital role in
causing hyperuricemia and gout (11). Allopurinol is a clinically
used XO inhibitor in the treatment of gout. However, because of
unwanted side effects of allopurinol, such as hepatitis, nephro-
pathy, and allergic reaction, research for new alternatives with
enhanced therapeutic activity and fewer side effects is necessary.
In addition, superoxide anion radicals generated by XO are
involved in various diseases, such as hepatitis, inflammation,
and carcinogenesis (12). Thus, the research for novel XO inhibi-
torswouldbe usefulnot onlyto treat gout but also totreat various
diseases associated with the increase of superoxide anion radicals.
As part of a continuing investigation on phloroglucinols
with anti-inflammatory and antioxidant activities, we report the
Phloroglucinols are a major class of secondary metabolites of
natural products. They have been investigated widely for its
biological activity (1). In previous papers, we isolated several
new terpenoids and phloroglucinols from Garcinia subelliptica
Merr. (Clusiaceae) and reported their anti-inflammatory activity,
cytotoxicity, and DNA strand-scission (2-4). However, phlo-
roglucinol derivatives isolated from Hypericum papuanum Ridley
(Clusiaceae) showed antioxidant activity (5). A phloroglucinol,
hyperielliptone HB isolated from Hypericum geminiflorum re-
vealed significant inhibition of oxidative DNA damage and an
inhibitory effect on xanthine oxidase (XO) activity (6).
Mast cells and neutrophils, stimulated with various inducers
may contribute to inflammatory disorders. Following the activa-
tion of macrophages, TNF-R and NO were generated in response
to lipopolysaccharide (LPS) (7, 8). NO plays a central role in
*Corresponding author. (S.C.Y.) E-mail: scyang@kmu.edu.tw.
(C.N.L.) Tel: þ 886 7 3121101. Fax: þ 886 7 5562365. E-mail:
lincna@cc.kmu.edu.tw.
pubs.acs.org/JAFC
Published on Web 09/15/2009
© 2009 American Chemical Society

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J. Agric. Food Chem., Vol. 57, No. 19, 2009 8783
Figure 1. Structures of phloroglucinols, garcinielliptones HA-HE (1-5), and C (6).
anti-inflammatory and antioxidant activities of phlorogucinols,
garcinielliptones HA-HE (1-5), and C (6) (Figure 1), isolated
form Garcinia subelliptica in the present article.
performed. This kinetics study was carried out in the absence and presence
of active compounds with varying concentrations of xanthine as substrate.
The initial rates were 0.5 and 3 min. The inhibition constant (Ki) was
determined from the slopes of the Lineweaver-Burk Plot for competitive
inhibition and intercept on vertical axis for noncompetitive inhibition (15).
Inhibition of Oxidative DNA Damage. A mixture of supercoiled
plasmid pBR322 DNA (1 μg/μL) and XA (2 mM)/XO (0.7 U/mL) in
10 mM phosphate buffer (pH 7.4) was incubated for 20 min with 500 μM
SOD, quercetin, and 1-6 in a total volume of 20 μL in a 1.5 mL
microcentrifuge tube at 37 ꢀC. Quercetin was used as the positive control.
After incubating for 20 min, a 15 μL aliquot of the mixture was loaded into
1.0% agarose gel containing ethidium bromide (0.05 μg/mL) in Tris-
acetate-ethylenediaminetetraacetic acid (EDTA) buffer. The electrophor-
esis 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 SOD, quercetin, and 1-6 served as the control. The gel electro-
phoretic mobility of the various forms of DNA was compared with the
control (16).
MATERIALS AND METHODS
Chemicals. Garcinielliptone HA-HE (1-5) and C (6) were isolated
fromGarcinia subelliptica (2,3). Compound48/80, histamine, formyl-Met-
Leu-Phe (fMLP), mepacrine, trifluoperazine, heparin, bovine serum
albumin, phenolphthalein-β-D-glucuronidase, O-phthadialdehyde, cyto-
chrome c, superoxide dismutase (type I, from bovine liver), bacterial LPS
(Escherichia coli, serotype O111: B4), and L-NAME were obtained from
Sigma-Alderich (St. Louis, MO). Hanks’ balanced salt solution (HBSS)
was obtained form Gibco Lab, Grand Island, NY. Dextran T 500 was
purchased from pharmacia LKB, Taipei, Taiwan. Dimethyl sulfoxide
(DMSO) was obtained from Merck. Cisplatin was obtained from Phar-
macia & Upjohn, Milan, Italy. All culture reagents were obtained from
Gibco BRL.
Mast Cell Degranulation, Neutrophil Degranulation, Superoxide
Anion Generation, Macrophage Culture and Drug Treatment, and
NO Determination. The inhibitory assays for the chemical mediator
induced by various stimulants in mast cells, neutrophils, and RAW 264.7
cells were performed by the methods as described in the literature (13).
Assay of XO Activity. The XO activity with xanthine as the substrate
was measured at 25 ꢀC, according to the protocol of Kong and others (14),
with modifications. The assay mixture consisting of 50 μL of test solution,
60 μL of 70 mM phosphate buffer (pH 7.5), and 30 μL of enzyme solution
(0.1 units/mL in 70 mM phosphate buffer (pH 7.5) was prepared immedi-
ately before use. After preincubation at 25 ꢀC for 15 min, the reaction was
initiated by the addition of 60 mL of substrate solution (150 μM xanthine in
the same buffer). The reaction was monitored for 5 min at 295 nm. The XO
activity was expressed as micromoles of uric acid per minute.
Cell Culture and MTT Assay for Cell Viability. NTUB1 human
eurthelial carcinoma cells were maintained in RPMI 1640 medium
supplemented with 10% fetal bovine serum (FBS), 100 unit/mL penicil-
lin-G, 100 μg/mL streptomycin, and 2 mM
L-glutamine. SV-HUC1
immortalized normal human urothelial cell line was obtained from
American type Culture Collection (Rockville, USA) and maintained in
F12 medium supplemented with 10% fetal bovine serum (FBS), 100 unit/
mL penicillin-G, 100 μg/mL streptomycin, and 2 mM
L-glutamine.
The cells were cultured at 37 ꢀC in a humidified atmosphere containing
5% CO2.
For evaluating the cytotoxic effect of 3 with cisplatin, a modified 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma
Chemical Co.) assay was performed (17). Briefly, the cells were plated
at a density of 1800 cells/well in 96-well plates and incubated at 37 ꢀC
overnight before drug exposure. Cells were then cultured in the presence
Lineweaver-Burk Plots. To determine the mode of inhibition by the
active compound from the plant, Lineweaver-Burk Plot analysis was
of graded concentrations of
3 with or without 5 μM cisplatin

8784 J. Agric. Food Chem., Vol. 57, No. 19, 2009
Lin et al.
(Pharmacia & Upjohn) at 37 ꢀC for 72 h. At the end of the culture period,
50 μL of MTT (2 mg/mL in PB) was added to each well and allowed to
react for 3 h. Following centrifugation of plates at 1000g for 10 min, media
were removed, and 150 μL DMSO was added to each well. The propor-
tions of surviving cells were determined by absorbance spectrometry at
540 nm using an MRX (DYNEXCO) microplate reader. The cell viability
was expressed as a percentage of the viable cells from the control culture
conditions. The IC50 values of each group were calculated by the median-
effect analysis and presented as the mean ( standard deviation (SD).
Statistical Analysis. Data were expressed as the means ( SD.
Statistical analyses were performed using the Bonferroni t-test method
after ANOVA for multigroup comparison and the Student’s t-test method
for two-group comparison. P < 0.05 was considered to be statistically
significant.
RESULTS AND DISCUSSION
The anti-inflammatory effect of 1-6 (Figure 1) was studied in
vitro for their inhibitory effect on chemical mediators released
from mast cells, neutrophils, and macrophages. Compound 6
showed significant inhibitory effect on the release of β-glucur-
onidase and lysozyme from rat neutrophils stimulated with
fMLP/CB in a concentration dependent manner with % inhibi-
tion of 48.5 ( 3.7 at 30 μM and IC50 value of 27.4 ( 5.8 μM,
respectively (Figure 2), while 1-5 had no significant inhibitory
effect on mast cell and neutrophil degranulation (data not
shown). Compound 6 with a bicycle [3. 3. 1] and previously
reported phloroglucinols, garcinielliptin oxide, and garciniellip-
tone F (4) with a bicycle [4. 3. 0] and [3. 3. 1] nonane moieties
exhibited significant inhibitory effects on the release of β-glucur-
oniase and lysozyme from rat neutrophil (Figure 2), while
phloroglucinols such 1-5 had no effect on the release of
β-glucuronidase and lysozyme from the rat neutrophils in re-
sponse to fMLP/CB. It indicated that phloroglucinol with a
bicyclo [3. 3. 1] or [4. 3. 0] nonane skeleton enhanced the
inhibitory effect on the release of chemical mediators from rat
neutrophil response to fMLP/CB. Compounds 3, 4, and 6 showed
significant inhibitory effects in a concentration-dependent man-
ner on superoxide anion formation in neutrophils stimulated with
fMLP/CB with IC50 values of 5.8 ( 4.1, 16.6 ( 2.2, and 11.5 (
0.6 μM, respectively (Figure 3), while having no significant
inhibitory effect on superoxide anion formation in neutrophil
response to phorbol myristate acetate (PMA) (data not shown).
This clearly indicated that methylation of C-19-OH such as 2
shows a slight inhibitory effect on superoxide anion formation in
neutrophil culture medium in response to PMA with a %
inhibition of 48.6 ( 12.1 at 3 μM and attenuated the inhibitory
effect on superoxide anion generation in neutrophil response to
fMLP/CB. But an R-oriented chemical bond between C-18 and
C-19 such as 3 enhanced the inhibitory effect. The observation
that 3, 4, and 6 had no appreciable effect on PMA-induced
response suggests the involvement of a PMA-independent signal-
ing pathway (18).
Figure 2. Inhibitory effects of 6 and TFP on the release of β-glucuronidase
and lysozyme from rat neutrophils stimulated with fMLP (1 μM)/CB (5 μg/
mL). Data are presented as the means ( SD (n = 3-6). Trifluoperazine
(TFP) was used as a positive control. * p < 0.01 compared to the control
value.
Figure 3. Inhibitory effects of 3, 4, 6, and DPI (diphenylene iodonium
chloride) on superoxide anion generation in rat neutrophils stimulated with
fMLP(0.3 μM)/CB (5μg/mL). Dataarepresentedasthe means( SD (n =
3-6). DPI was used as the positive control. * p < 0.05 and ** p < 0.01
compared to the control value.
The antioxidant activities of 1-6 and allopurinol (positive
control), a drug clinically prescribed in the clinic for gout
treatment, on xanthine oxidase (XO) were studied in vitro. As
shown in Figure 5, compounds 1, 3-6, and allopurinol inhibited
XO activity in a concentration-dependent manner with IC50
values of 31.7 ( 0.4, 16.6 ( 0.2, 172.6 ( 8.9, 104.3 ( 1.9,
59.9 ( 1.1, and 2.0 ( 0.7 μM, respectively. Compound 3 was
identified as a stronger XO inhibitor than those of the same
chemical skeleton such as 2 and 4. It indicated that a free OH
group attached at C-19 and the R-oriented chemical bond
between C-18 and C-19 also enhanced the activity of XO inhibi-
tion. For xanthine oxidase assays performed as a function of
different concentrations of xanthine in the absence or presence of
1 and 3, respectively, Lineweaver-Burk transformation of the
data are shown in Figure 6. The data indicate that 1 and 3 act as
noncompetitive and mixed inhibitors of xanthine oxidase, respec-
tively. It revealed that 1 could be bound to free enzyme but that 3
could be bound to free enzyme and the enzyme-substrate
TNF-R is an important pro-inflammatory cytokine with im-
mune and inflammatory functions and is generally considered as
principal mediator of septic shock. As shown in Figure 4, com-
pounds 1, 5, and the positive control, genistein, revealed potent
inhibitory effects on TNF-R formation in RAW 264.7 cells
stimulated with LPS in a concentration-dependent manner
with IC50 values of 4.8 ( 1.9, 23.7 ( 3.0, and 18.7 ( 3.6 μM,
respectively while 2-4 and 6 did not reveal an inhibitory effect on
TNF-R formation. Compound 1, exhibited stronger inhibitory
effect than the positive control, genistein, and may be used for
septic shock. The results showed that phloroglucinol with two
furan rings or a pyran and furan rings fused on a benzene ring
significantly enhanced the inhibitory effect on the formation of
TNF-R inthe culture mediumofRAW 264.7 cell responsetoLPS.

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J. Agric. Food Chem., Vol. 57, No. 19, 2009 8785
Figure 7. Inhibition of DNA strand breaks induced by O-2 • (generated by
XA/XO) in the presence of 1 and 3-6 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, control; lane 3, control þ SOD (300 μM);
lane 4, control þ quercetin (300 μM) serving as the positive control;lane 5,
control þ 1 (300 μM); lane 6, control þ 3 (300 μM); lane 7, control þ 4
(300 μM); lane 8, control þ 5 (300 μM); lane 9, control þ 6 (300 μM).
Figure 4. Inhibitory effects of 1, 5, andgenisteinonthe formation ofTNF-R
in the culture media of RAW 264.7 cells in response to LPS (1 μg/mL).
Data are presented as the means ( SD (n = 3-6). Genistein was used as
the positive control. * p < 0.05 compared to the control value.
Figure 8. Compound 3 (A) or 3 cotreated with cisplatin (B) induced
NTUB1 cell death. Cell viability was assessed by the MTT assay 72 h after
treating by different concentrations of 3 or 5 μM cisplatin cotreated with
different concentrations of 3. * p < 0.05, ** p < 0.01, and *** p < 0.001
compared to the control value.
Figure 5. Dose-dependent inhibition of XO by 1, 3-6, and allopurinol.
Data are presented as the means ( SEM, n = 3-6. * p < 0.05, ** p < 0.01,
and *** p < 0.001 compared to the control value.
peptide chain of the enzyme (19). Therefore, in addition to the
chemical skeleton and conformation of the compound, the phenolic
hydroxyl, alcoholic hydroxyl, and carbonyl may interfere with
interaction between the enzyme and enzyme-substrate complex.
ROS have been known to damage many biological macro-
molecules, with DNA being a significant target. The ability of
1-6 to inhibit the DNA damage caused by O-2 •generated by XA/
XO) was investigated by the agarose gel electrophoresis method.
As shown in Figure 7, lane 1 served as the control and has two
different plasmid DNA forms, open circular and supercoiled
DNA. However, the plasmid DNA was damaged in the presence
of XA/XO (lane 2) with an increased proportion of the open
circular form. Adding SOD (500 μM) to the XA/XO system
inhibited the DNA damage completely (lane 3), confirming the
role of O-2 • in plasmid DNA damage. Quercetin and phloroglu-
cinols 1 and 3-6 at the same treated concentration, 300 μM,
significantly inhibited DNA scission (lane 4-9). The above result
revealed that these compounds may inhibit the formation of ROS
in cells and may protect cell death (20).
Figure 6. Linweaver-Burk plot of XO inhibition of 1 (A) and 3 (B) with
various concentrations of xanthine. Linweaver-Burk transformed data
were plotted and followed by line regression of the points. Data represent
the average of triplicate experiments.
For further evaluation of the protective effect on cells, NTUB1
or SV-HUC1 immortalized normal human urothelial cells were
treated with various concentrations of selective compound 3,
showing a potent inhibitory effect on the chemical mediator and
XO, in the presence and absence of 5 μM cisplatin, and we
complex. Biochemically, enzyme inhibition is associated with
hydrogen binding of phenolic hydroxyls and/or carbonyls of the
substrate with the amide carbonyls and/or amino groups in the

8786 J. Agric. Food Chem., Vol. 57, No. 19, 2009
Lin et al.
Figure 9. Cisplatin cotreated with 3 in SV-HUC1 cells. Cell viability was assessed by the MTT assay 72 h after treating with different concentrations of 3 and
5 μM cisplatin. *p < 0.05 compared to the control value.
Figure 10. Effect of 3 and 3 cotreated with cisplatin in the production of ROS in NTUB1 cells. (A) Control; (B) 20 μM cisplatin; (C) 20 μM 3; (D) 50 μM 3;
(E) 20μM cisplatinþ 20 μM3;(F) 20μMcisplatinþ 50 μM 3, and20μMcisplatincotreated with differentconcentrationsof3for24h, andtheamountofROS
was assayed by H2DCFDA staining. Results were repeated by independent experiments.
determined the cell viabilities by the MTT assay. Compound 3 at
0.3 to 30 μM or 3 to 60 μM did significantly cause increased
NTUB1 (Figure 8) or SV-HUC1 (data not shown) cell death in a
concentration-dependent manner. Further evaluations showed
that the viability of NTUB1 cells treated with 3 and 5 μM cisplatin
was protected by 3 between 6 to 60 μM, while at 100 μM, it did
not protect NTUB1 cells from cisplatin-induced cytotoxicity
(Figure 8). Compound 3 exhibited weak cytotoxicity against
SV-HUC1 cells (data not shown), while at 15 to 60 μM cotreated
with 5 μM cisplatin, it significantly protected SV-HUC1 cell
death induced by cisplatin (Figure 9).
Many studies have suggested that cisplatin stimulates cells to
produce ROS (21). ROS have been shown as causative factors
involved in many human degenerative diseases, and antioxidants
have been found to have some degree of preventive and thera-
peutic effects on these disorders (22). Compounds 1, 3, and 6
significantly inhibited the DNA damage induced by O-2 • in vitro
and also the XO activity. This suggested that each of the three
compounds or each of these compounds cotreated with cisplatin
may inhibit the formation of ROS through the inhibitory effect
on XO and protected cells through the diminishing offree radicals
in NTUB1 and SV-HUC1 cells, respectively.
Now, it is clear that ROS have a cell signaling role in many
biological systems, both in animals and plants. ROS induce
programmed cell death or necrosis, induced or suppressed the
expression of many genes, and activated cell signaling cascades,
such as those involving mitogen-activated protein kinases (20).
As shown in Figure 10, the amount of ROS produced by 3
cotreated with cisplatin in NTUB1 cells were less than that
generated by cisplatin or 3 alone as determined by the fluorescent
dye, H2DCFDA, which preferentially detected intracellular ROS.
The results showedthat3 mediatesthrough its inhibitory effect on

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J. Agric. Food Chem., Vol. 57, No. 19, 2009 8787
XO and protected cells through the diminishing of ROS in
NTUB1 cells in the presence of cisplatin, and prevents the
following cell death. It also suggested that 3 may mediate through
its inhibitory effect on XO and may protect cells through the
diminishing in immortalized normal human urothelial cells
(SV-HUC1) in the presence of cisplatin and prevent the following
cell death.
In conclusion, the phloroglucinols showed significant anti-
inflammatory and antioxidant activities. The protective effect of3
cotreated with cisplatin is due to its significant inhibitory effect on
XO activity and diminishing the formation of free radicals. Our
findings suggested that these phloroglucinols may protect against
radical stress by inhibiting ROS formation and may be used as a
protective agent for diseases associated with increased amounts of
ROS. It is valuable for further studies on the protective effect and
the bioavailability of selective compound 3 in vivo.
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Received March 19, 2009. Revised manuscript received July 15, 2009.
Accepted August 26, 2009. This work was supported by a grant from the
National Science council of Republic of China (NSC 96-2628-B-037-
012) and partially supported by a fund of KMU-EM-97-2.1.b.
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