Chemical studies on antioxidant mechanism of garcinol: Analysis of radical reaction products of garcinol with peroxyl radicals and their antitumor activities

Article abstract of DOI:10.1016/S0040-4020(02)01411-4

Antioxidant actions of garcinol (1), a polyisoprenylated benzophenone, purified from Garcinia indica fruit rind, are believed to contribute to its chemopreventive activity. However, the mechanisms of its antioxidant reactions remain unclear. The objective of this study was to characterize the reaction products of garcinol with peroxyl radicals generated by thermolysis of the azo initiator azo-bis-isobutyrylnitrile (AIBN). Structure elucidation of these products can provide insights into specific mechanisms of antioxidant reactions. Four reaction products (2-5) were isolated and identified. Their structures were determined on the basis of detailed high field 1D and 2D spectral analysis. The identification of these products provides the first unambiguous proof that the double bond of the isopentenyl group is a principal site of the antioxidant reaction of 1. The induction of apoptosis in human leukemia HL-60 cells, the inhibition of NO generation, and the inhibition of LPS-induced iNOS gene expression by Western blot analysis by 1 and its four oxidation products (2-5) were investigated.

Full text of DOI:10.1016/S0040-4020(02)01411-4

Tetrahedron 58 (2002) 10095–10102
Chemical studies on antioxidant mechanism of garcinol:
analysis of radical reaction products of garcinol with peroxyl
radicals and their antitumor activities
a
Shengmin Sang, Chiung-Ho Liao, Min-Hsiung Pan, Robert T. Rosen, Shoei-Yn Lin-Shiau,
b
b
a
b
b
Jen-Kun Lin and Chi-Tang Ho
b,
*
a
Department of Food Science and Center for Advanced Food Technology, Rutgers University,
65 Dudley Road, New Brunswick, NJ 08901-8520, USA
Institute of Biochemistry and Toxicology, College of Medicine, National Taiwan University, 1 Section 1, Jen-ai Road, Taipei, Taiwan, ROC
b
Received 10 July 2002; revised 30 October 2002; accepted 31 October 2002
Abstract—Antioxidant actions of garcinol (1), a polyisoprenylated benzophenone, purified from Garcinia indica fruit rind, are believed to
contribute to its chemopreventive activity. However, the mechanisms of its antioxidant reactions remain unclear. The objective of this study
was to characterize the reaction products of garcinol with peroxyl radicals generated by thermolysis of the azo initiator azo-bis-
isobutyrylnitrile (AIBN). Structure elucidation of these products can provide insights into specific mechanisms of antioxidant reactions. Four
reaction products (2–5) were isolated and identified. Their structures were determined on the basis of detailed high field 1D and 2D spectral
analysis. The identification of these products provides the first unambiguous proof that the double bond of the isopentenyl group is a principal
site of the antioxidant reaction of 1. The induction of apoptosis in human leukemia HL-60 cells, the inhibition of NO generation, and the
inhibition of LPS-induced iNOS gene expression by Western blot analysis by 1 and its four oxidation products (2–5) were
investigated. q 2002 Elsevier Science Ltd. All rights reserved.
6
1
. Introduction
almost the same extent as DL-a-tocopherol by weight; in
a phenazine methosulfate/NADH-nitroblue tetrazolium
system, garcinol exhibited superoxide anion scavenging
activity and suppressed protein glycation in a bovine serum
Garcinol (1), also called camboginol, is a polyisoprenylated
benzophenone derivative isolated from Garcinia indica
1
–4
9
and other species.
The dried rind of G. indica (cv.
albumin/fructose system. It also showed nearly three times
greater 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical
scavenging activity than DL-a-tocopherol by weight.
However, the antioxidant mechanisms of garcinol remain
unclear. We recently reported the structures of two major
oxidation products (4 and 5) of garcinol with DPPH. The
identification of these structures provided the first unam-
biguous evidence that the principal oxidation sites of
garcinol are on the 1,3-diketone and the phenolic ring
part. It is generally accepted that depending on how
oxidation is achieved, the condensation products are
different. To continue our study on the antioxidant
mechanisms of garcinol, we reacted garcinol with peroxyl
Kokum) is used as a garnish for curry and in some of the
folklore medicine in India and contains 2–3% garcinol by
9
1
,2
weight. Garcinol is structurally similar to a well-known
antioxidant, curcumin (6), which contains both phenolic
hydroxyl groups and an enol form of a b-diketone moiety.
Recently, garcinol has attracted considerable interest
because of its associated beneficial health properties,
1
0
5
6
including antibiotic activities, antiulcer activity, sup-
7
pressed colonic aberrant crypt foci (ACF) formation, and
induction of apoptosis through cytochrome c release and
8
activation of caspases in human leukemia HL-60 cells. It
also showed strong antioxidant activity. In the H O /NaOH/
2
2
0
DMSO system, garcinol suppressed superoxide anion,
hydroxyl radical, and methyl radical; in the Fenton reaction
system, garcinol suppressed hydroxyl radical more strongly
than DL-a-tocopherol; in the hypoxanthine/xanthine oxidase
system, emulsified garcinol suppressed superoxide anion to
radicals generated by thermolysis of the initiator 2,2 -azo-
bis-isobutyronitrile (AIBN). The antioxidant process of
this reaction is thought to be divided into the following
stages:
1. Radical generation stage
K
Keywords: garcinol; peroxyl radical; antioxidant mechanism; antitumor
activities.
z
RNvNR ! 2R þ N
2
*
Corresponding author. Tel.: þ732-932-9611x235; fax: þ732-932-6776;
z
z
2
R þ 2O ! 2ROO
e-mail: ho@aesop.rutgers.edu
2
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01411-4

10096
S. Sang et al. / Tetrahedron 58 (2002) 10095–10102
Figure 1. Structures of compounds 1–5 and curcumin (6).
antioxidant mechanism studies. In the study presented here,
we have succeeded in isolating and characterizing reaction
products of garcinol with alkylperoxyl radicals from AIBN
in a homogeneous acetone system. Four reaction products
2
3
. Radical trapping stage
z
z
ROO þ AH O ROOH þ A
(2–5) were isolated and identified. Their structures were
determined on the basis of detailed high field 1D and 2D
spectral analysis. The induction of apoptosis in human
leukemia HL-60 cells, the inhibition of NO generation, and
the inhibition of LPS-induced iNOS gene expression by
Western blot analysis by 1 and its four oxidant products
. Radical termination stage
z
z
A þ X ! nonradical material
(
2–5), and curcumin (6) were investigated.
AIBN decomposed thermally to yield alkyl radicals (Rz),
which react with oxygen rapidly to generate peroxyl radicals
z
z
(
radical, and X is another radical species or the same species
ROO ). AH is the phenolic antioxidant, A is the antioxidant
z
2. Results and discussion
z 11
as A . Although the second stage is a reversible process,
2.1. Structural elucidation of four oxidation products
(2–5)
the third stage is irreversible and produces stable radical
termination compounds. Structural information about these
nonradical products would afford important contributions to
Reaction of garcinol (1) with peroxyl radical was carried out

S. Sang et al. / Tetrahedron 58 (2002) 10095–10102
10097
and four major oxidation products (2–5) were isolated and
identified on the basis of their spectral data (Fig. 1).
respectively. All of these indicated that the carbonyl at C-3
was enolized and the oxygen was attached to C-19. The
proton and carbon shifts at position 18 (d 92.1, d 4.42)
C
H
Compound 2, a yellow amorphous solid, was assigned the
molecular formula of C H O determined by HRFAB-MS
were deshielded relative to those of a secondary alcohol and,
when considered with the molecular formula, suggested
12,13
that a hydroperoxyl group was present at this position.
3
þ
8 50 8
m/z 635.3581 [MþH] (calcd for C H O , 635.3584), as
3
8 51 8
1
3
well as from its C NMR data. The molecular formula
indicated fourteen degrees of unsaturation, which showed
Compound 2 gave a positive peroxide reaction with
1
4
FeSCN. The a configuration for this hydroperoxyl group
was supported by the large J17,19 coupling constant (dd,
J¼7.2, 9.6 Hz). Thus, the structure of 2 was deduced as
shown (Fig. 1). The complete interpretation of the NMR
data was based on the results of COSY, TOCSY, HMQC
and HMBC.
1
that 2 had the same unsaturation as 1. The H NMR
spectrum of 2 showed the presence of three AMX pattern
aromatic protons at d 7.39 (d, J¼1.8 Hz), 7.26 (dd, J¼1.8,
8
oxygenated methine at d 4.42 ppm; two isopentenyl groups
.4 Hz), and 6.79 (d, J¼8.4 Hz) ppm, respectively; one
[
two vinylic protons at d 4.97, t, J¼6.0 Hz, and 4.87, t,
J¼6.0 Hz; and four vinylic methyl groups at d 1.64 (for two
methyl signals), 1.59, and 1.57 ppm, respectively], one
isopropenyl group [two singlets of 2H at d 4.48 and 4.44,
together with a methyl singlet at d 1.44], and four methyl
groups on saturated carbons [four methyl singlets at d 1.15,
Compound 3, a yellow amorphous solid, had a molecular
formula of C H O determined by APCIMS (m/z
3
8
50
6
þ
same as the molecular formula of 1. The H NMR spectrum
13
[MþH] 603) as well as its C NMR data, which was the
1
of 3 also showed the presence of three AMX pattern
aromatic protons at d 7.42 (d, J¼1.8 Hz), 7.00 (dd, J¼1.8,
8.4 Hz), and 6.66 (d, J¼8.4 Hz) ppm, respectively; three
isopentenyl groups [three vinylic protons at d 5.10, t,
J¼6.0 Hz, 4.90, t, J¼6.0 Hz, and 4.85, t, J¼6.0 Hz; and five
singlet methyl groups at d 1.71, 1.65, 1.62, and 1.56 (for two
methyl groups), respectively], and four methyl groups on
saturated carbons [four methyl singlets at d 1.22, 1.15, 0.97
and 0.94], in addition to methylene and methine protons
[a complex multiplet of 12H in the region of d 3.10–1.45].
1
protons [a complex multiplet of 12H in the region of d
.09, 1.08 and 1.04], in addition to vinylic and methine
1
2
.80–1.45]. Thus the significant difference in the H NMR
spectrum of 2 compared to 1 was the absence of the double
bond of one isopentenyl group. As mentioned above, 2 had
the same unsaturation as 1. All of these suggested that there
was one more ring in 2 than in 1. The above findings were in
1
3
agreement with the C NMR spectrum of 2. It also showed
the presence of two isopentenyl groups [two methine
carbons of trisubstituted olefinic groups at d 124.5 and
1
Thus, the significant difference in the H NMR spectrum of
1
2
23.3, and four methyl groups at d 18.1, 18.2, 25.9, and
6.0], one isopropenyl group [d 112.9 for a terminal
3 compared to that of 1 was the absence of the double bond
of the isopropenyl group. As mentioned above, 3 had the
same molecular weight as 1. This implied that there was one
more ring in 3 than in 1. The above findings were in
methylene carbon, and d 18.0 for a methyl signal], and
four methyl groups on saturated carbons [d 23.9, 24.4, 26.5
and 27.3]; three methine carbons for the aromatic ring at d
1
3
agreement with the C NMR spectrum of 3. It also showed
the presence of three isopentenyl groups [three methine
carbons of trisubstituted olefinic groups at d 125.3, 124.9
and 123.9, and six methyl groups at d 17.7, 17.8, 17.9, 25.4,
25.5, and 25.7], and four methyl groups on saturated carbons
[d 21.1, 22.3, 26.6, and 28.2]; three methine carbons for the
aromatic ring at d 114.6, 119.7 and 121.4; two oxygen-
substituted aromatic carbons at d 144.9 and 150.8; and one
oxygenated quaternary carbon at d 86.8. Furthermore, in the
1
14.8, 116.8 and 124.3; two oxygen-substituted aromatic
carbons at d 143.8 and 150.5; one oxygenated quaternary
carbon at d 71.6, and one oxygenated methine at d 92.1.
Furthermore, in the C spectral data one of the carbon
atoms of the enolized 1,3-diketone in 1 was changed from
d194.0 (or 195.2) to d 175.2 in 2. The C spectral data of
the carbonyl (C-10) was also changed from d 199.1 in 1 to d
1
3
1
3
1
90.6 in 2. The HMBC correlation between Cd175.2 and
1
3
H-17 (d 2.68 and 2.07), Cd194.2 and H-7 (d 2.19 and 2.04),
H-29 (d 2.08 and 1.74); Cd190.6 and H-12 (d 7.39), H-16 (d
C spectral data, the chemical shift of one of the carbon
atoms of the enolized 1,3-diketone in 1 was changed from d
1
3
7
.26); Cd71.6 and H-17 (d 2.68 and 2.07), H-20 (d 1.04),
194.0 (or 195.2) to d 171.9 in 3. The C spectral data of the
carbonyl (C-10) was also changed from d 199.1 in 1 to d
194.7 in 3. All of these indicated that the carbonyl at C-1
was enolized and the oxygen was attached to C-31.
H-21 (d 1.09) and Hd92.1 (d 4.42); Cd92.1 and H-17 (d 2.68
and 2.07), H-20 (d 1.04) and H-21 (d 1.09) (Fig. 2)
suggested that d 175.2 could be assigned to C-3, d 194.2 to
C-1, d 190.6 to C-10, d 71.6 to C-19, and d 92.1 to C-18,
1
,2,15
Therefore, compound 3 was identified as cambogin.
This was further confirmed by comparing 3 with standard
cambogin by TLC plate.
Compound 4, a yellow amorphous solid, was assigned the
molecular formula of C H O determined by positive-ion
3
8 48 6
þ
NMR data. The molecular formula indicated fifteen degrees
13
APCI-MS ([MþH] at m/z 601), as well as from its
C
of unsaturation, which showed that 4 had one more
1
13
unsaturation than 1. The H and C NMR data of 4 are
identical with those of GDPPH-1 that we reported from the
1
0
reaction between garcinol and the stable radical DPPH.
Thus, compound 4 was identified as shown (Fig. 1). This
was further confirmed by comparing 4 with standard
GDPPH-1 by TLC plate.
Figure 2. Significant HMBC (H!C) correlations of 2.

10098
S. Sang et al. / Tetrahedron 58 (2002) 10095–10102
Figure 3. Induction of apoptosis by compounds 1–5, and curcumin (6) in HL-60 cells. HL-60 cell was treated with different concentrations (2.5, 5, 10, and
20 mM) of compounds 1–5, and curcumin for 24 h and apoptosis was quantified by flow cytometry. The method of flow cytometry used is described in Section
3.
Compound 5 was isolated as a yellow amorphous solid. The
positive-ion APCI-MS of 5 displayed a molecular ion peak
2.2. Induction of apoptosis by compounds 1–5, and
curcumin (6) in human leukemia HL-60 cells
þ
C H O , which was the same as that of 4. The H and C
at m/z [MþH] 601, supporting a molecular formula of
1
1
Physiological cell death is characterized by apoptotic
morphology, including chromatin condensation, membrane
blebbing, internucleosomal degradation of DNA, and
apoptotic body formation. In each case, nucleosomal DNA
ladders, which are typical of apoptosis, were visible on
agarose gel after staining with ethidium bromide. A sub-G1
3
8 48 6
NMR data of 5 are identical with those of GDPPH-2 that we
reported from the reaction between garcinol and the stable
1
0
radical DPPH. Thus, compound 5 was identified as shown
Fig. 1). This was further confirmed by comparing 5 with
standard GDPPH-2 by TLC plate.
(
Figure 4. Effects of compounds 1–5, and curcumin (6) on LPS-induced nitrite production in RAW 264.7 cells. The cells were treated with different
concentrations of compounds and LPS (50 ng/mL) for 16 h. Nitrite was determined by Griess reaction, as described in Section 3.

S. Sang et al. / Tetrahedron 58 (2002) 10095–10102
10099
Figure 5. Western blot analysis of the inhibition of LPS-induced iNOS protein expression by compounds 1–5, and curcumin (6). RAW 264.7 were co-treated
with 50 ng/mL of LPS and 2.5 mM of compounds 1–5, and curcumin for 24 h. Total protein was isolated for western blot analysis of iNOS and b-actin.
Quantification of band intensities was via three independent experimental results by densitometry (IS-1000 Digital Imaging System).
(
sub-2N) DNA peak, which has been suggested to be the
inhibited iNOS protein (Fig. 5). The inhibitory potency
was as follows: garcinol.5.3.4.2.curcumin, at 10 mM.
The amount of b-actin protein as an internal control
remained unchanged.
apoptotic DNA was detected in cells that were treated with
compounds 1–5, and curcumin (6), and stained by
propidium iodide. As shown in Fig. 3 the percentage of
apoptotic HL-60 cells were 72.06, 4.27, 70.23, 67.6, 60.35,
and 11.12% after 18 h of incubation with compounds 1–5,
and curcumin (20 mM), respectively. Among them, garcinol
and compounds 3–5 appeared to be more potent and dose-
dependent on the induction of cell apoptosis. When the
HL-60 cells were treated with the same concentration
2.5. Discussion
The purpose of this investigation was to isolate and
characterize the reaction products of garcinol with alkyl-
peroxyl radicals derived from AIBN in a homogeneous
solution. Our previous identification of garcinol oxidation
(
20 mM) of these compounds, the apoptotic potency was the
same. These data are consistent with DNA fragmentation
data not shown).
1
0
products 4 and 5 provided the first unambiguous evidence
that antioxidant reactions of garcinol with stable free radical
DPPH involve the 1,3-diketone and phenolic ring moieties.
In this current study, we identified two more reaction
products (products 2 and 3), which showed that the double
bond of the isoprenyl group was also a principal site of the
antioxidant reaction of garcinol. Compound 2 is a new
hydroperoxy derivative of garcinol. A similar compound
(
2.3. Inhibition of NO generation by compounds 1–5, and
curcumin (6)
Compounds 1–5, and curcumin were examined to deter-
mine whether they affect NO production in macrophages
activated with LPS for 16 h. Of these compounds, garcinol
inhibited LPS-stimulated NO generation most strongly;
however, compounds 1–5, and curcumin all markedly
reduced NO generation in a concentration-dependent
manner (Fig. 4). The inhibitory potency was estimated as
follows: garcinol.4.5.curcumin.3.2, at 10 mM.
Inhibition of NO production was not due to cytotoxicity,
as determined with trypan blue exclusion assay.
1
3
has been reported as a new natural product. Compound 3
was identified as cambogin, also named isogarcinol, which
has been reported from G. indica, G. cambogia, and other
1
,2,4,15,16
species.
However, ours is the first study to report
the formation of cambogin as an oxidation product
during the antioxidant reaction of garcinol. Cambogin has
biological activities that are similar to those of garcinol. It
has been claimed for use as hyaluronidase inhibitor for
prevention of skin aging and as antiinflammatory and
1
7
2
.4. Inhibition of LPS-induced iNOS gene expression by
Western blot analysis by compounds 1–5, and curcumin
6)
antitumor agents, as a lipase inhibitor, an anti-obesity
1
8
agent, and a hypolipidemic, as an inhibitor for Epstein–
Barr virus early antigen induction and as an antitumor
(
1
9
20
agent, and as an antiulcer agent. Cambogin has also been
reported for control of methicillin-resistant Staphylococcus
aureus.
Compounds 1–5, and curcumin were examined to deter-
mine whether they affect iNOS protein in macrophages
activated with LPS (100 ng/mL) for 16 h. RAW 264.7 cells
were treated. Inhibition of iNOS protein by these com-
pounds was detected at 2.5, 5, 10, 20 mM. RAW 264.7 cells
did not express iNOS protein when incubated in the medium
without LPS for 16 h. Upon LPS treatment, iNOS protein
drastically increased in these cells, and co-treatment of cells
with LPS and the indicated compounds for 16 h significantly
From our elucidation of the chemical structures of these four
compounds, we proposed the antioxidant mechanism of
garcinol as shown in Fig. 6. As shown in Fig. 6, garcinol has
been proposed to react with peroxyl radicals by a single
electron transfer followed by deprotonation from the
hydroxyl group of the enolized 1,3-diketone to form a

10100
S. Sang et al. / Tetrahedron 58 (2002) 10095–10102
Figure 6. Proposed mechanism for the formation of oxidation products 2–5.
2
1,22
resonance pair.
If reaction was initiated at the hydroxyl
It is notable that compounds 4 and 5, the two major reaction
products of our two model oxidation systems (DPPH system
and peroxyl radical system), showed similar inhibitory
effects on cell viability in human leukemia HL-60 cells as
garcinol. Our future work will focus on whether these
two reaction products of garcinol can show similar or even
stronger activities in other biological systems, such as
anti-inflammation, and antiulcer activities.
group of C-3, 2 and 4 would be formed, and 3 and 5 would
be formed if reaction was initiated at the hydroxyl group of
C-1.
Cancer preventive effects have often been attributed to
2
2–27
antioxidant actions.
In order to understand whether
garcinol or its oxidation products play a functional role in
biological system, the antitumor activities of garcinol and
its four reaction products were individually tested. The
activities of these compounds were compared with curcu-
min, a well-known antioxidant. These included an antitumor
test of garcinol and its four reaction products (2–5)
and curcumin on induction of apoptosis in human leukemia
HL-60 cells, the inhibition of NO generation, and the
inhibition of LPS-induced iNOS gene expression by
Western blot analysis. These tests indicated that like 1,
compounds 3–5 showed strong inhibitory effects on these
assays. Compound 2 showed very weak activity. Garcinol,
and compounds 4 and 5 showed better inhibitory effects in
these assays than curcumin. The potency of these com-
pounds in apotosis-induction may vary with the different
cell lines. These findings might suggest possible chemo-
preventive ability of garcinol and its oxidation products.
Analysis of these products could provide a unique tool for
assessing the contribution of antioxidant reactions to the
disease preventive effects of garcinol.
3. Experimental
3.1. General procedure
1
1
H (600 MHz), C (150 MHz) and all 2D NMR spectra were
run on a Varian AM-600 NMR spectrometer, with TMS as
internal standard. FT-IR was performed on a Magna 550
spectrometer. The APCI MS was performed on a Fisons/VG
Platform II mass spectrometer. HREI MS was run on JEOL
AX-505 double focusing mass spectrometer. Thin-layer
chromatography was performed on Sigma-Aldrich TLC
plates (250 mm thickness, 2–25 mm particle size), with
compounds visualized by spraying with 5% (v/v) H SO in
2
4
ethanol solution. CD OD was purchased from Aldrich
3
Chemical Co. Ferrous ammonium sulfate and ammonium
thiocyanate was purchased from Sigma Chemical Co. AIBN
1
was purchased from Sigma Chemical Co.: H NMR (CDCl ,
3

S. Sang et al. / Tetrahedron 58 (2002) 10095–10102
10101
1
3
2
6
00 MHz): d 1.66 (s, CH3); C NMR (CDCl , 150 MHz): d
3
negative APCI-MS m/z 633 [M2H] , HRFAB-MS m/z
þ
25.3 (q, CH ), 68.3 (s, C), 119.3 (s, CuN). Garcinol was
0
isolated from G. indica dried fruit rind.
635.3581 [MþH] (calcd for C H O , 635.3584).
38 51 8
3
1
Compound 3 was isolated as a pale amorphous substance:
1
3
2
.1.1. Oxidation of 1 and isolation of reaction products
–5. Garcinol (1) (2.0 g, 3.34 mmol) was allowed to react
H NMR (CD OD, 600 MHz): d 7.42 (d, J¼1.8 Hz), 7.00
3
(dd, J¼1.8, 8.4 Hz), 6.79 (d, J¼8.4 Hz), 6.66 (d, J¼8.4 Hz),
5.10 (t, J¼6.0 Hz), 4.90 (t, J¼6.0 Hz), 4.85 (t, J¼6.0 Hz), at
d 1.71 (s), 1.65 (s), 1.62 (s), 1.56 (s), 1.22 (s), 1.15 (s), 0.97
with AIBN (6.0 g, 15.7 mmol) in 50 mL acetone incubated
at 508C for 12 h. After evaporation of the solvent in vacuo,
the residue was first applied to silica gel column eluting with
chloroform to get rid of the AIBN and 1 (400 mg) and then
with methanol to get a mixture of four reaction products.
The mixture was subjected to RP-C18 silica gel column
eluting by a 90% methanol–water solvent system to give
1
3
(s) and 0.94 (s); C NMR (CD OD, 150 MHz): see Table 1;
3
2
negative APCI-MS m/z 601 [M2H] .
Compound 4 was isolated as a pale amorphous substance:
13
1
H NMR (CD OD, 600 MHz) and C NMR (CD OD,
3
3
1
0
4
4
0 mg compound 2, 30 mg compound 3, 200 mg compound
and 100 mg compound 5.
150 MHz): the same as GDPPH-1; positive APCI-MS m/z
601 [MþH]þ.
Compound 2 was isolated as a pale amorphous substance:
1
Compound 5 was isolated as a pale amorphous substance:
1
13
H NMR (CD OD, 600 MHz): d 7.39 (d, J¼1.8 Hz), 7.26
H NMR (CD OD, 600 MHz) and C NMR (CD OD,
3 3
3
1
0
(
4
dd, J¼1.8, 8.4 Hz), 6.79 (d, J¼8.4 Hz), 4.97 (t, J¼6.0 Hz),
150 MHz): the same as GDPPH-1; positive APCI-MS m/z
601 [MþH]þ.
.87 (t, J¼6.0 Hz), 4.48 (s), 4.44 (s), 4.42 (dd, J¼7.2,
9.6 Hz), 1.64 (s), 1.59 (s), 1.44 (s), 1.15 (s), 1.09 (s), 1.08 (s)
and 1.04 (s); C NMR (CD OD, 150 MHz): see Table 1;
1
3
3.2. Preparation of ferrous thiocyanate reagent
3
C
Table 1. d (150 MHz) NMR spectral data of compounds 1–3 (d in ppm, J
in Hz)
Ferrous ammonium sulphate (0.7 g) was dissolved in 10 mL
of a solution of ammonium thiocyanate (5 g) and a
14
concentrated sulfuric acid (1 mL) in water (100 mL).
a
1
a
2
b
3
This reagent was prepared just before use.
1
2
3
4
5
6
7
8
194.0 s
116.0 s
195.2 s
69.9 s
49.8 s
47.0 d
42.7 t
194.2 s
117.2 s
175.2 s
67.6 s
47.6 s
45.9 d
41.9 t
171.9 s
114.4 s
193.3 s
68.3 s
46.2 s
46.1 d
42.8 t
3
.3. Cell culture and chemicals
Human promyelocytic leukemia (HL-60) cells obtained
from American Type Culture Collection (Rockville, MD)
were grown in 90% RPMI 1640 and 10% fetal bovine serum
58.1 s
60.7 s
51.2 s
(GIBCO BRL, Grand Island, NY), supplemented with
9
207.1 s
199.1 s
127.8 s
116.6 d
143.9 s
149.9 s
114.4 d
120.2 d
27.2 t
207.7 s
190.6 s
129.9 s
116.8 d
143.8 s
150.5 s
114.8 d
124.3 s
26.7 t
207.3 s
194.7 s
129.9 s
119.7 d
144.9 s
150.8 s
114.6 d
121.4 s
25.8 t
2 mM glutamine (GIBCO BRL), 1% penicillin/strepto-
mycin (10,000 units of penicillin/mL and 10 mg/mL
streptomycin). RAW 264.7 cells were cultured in RPMI-
1640 (without phenol red) supplement with 10% endotoxin-
free heat-inactivated fetal calf serum (GIBCO, Grand
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
Island, NY). Lipopolysaccharide (LPS) (Escherichia coli
0127: B8) was purchased from Sigma Chemical Co.
122.8 d
92.1 d
123.8 d
3
.4. Flow cytometry
1
2
2
2
2
2
2
2
2
2
2
9
0
1
2
3
4
5
6
7
8
9
135.5 s
26.2 q
18.4 q
22.9 q
27.2 q
29.1 t
123.9 d
133.1 s
25.9 q
18.1 q
36.3 t
71.6 s
26.5 q
23.9 q
24.4 q
27.3 q
29.8 t
124.5 d
133.9 s
25.9 q
18.2 q
36.3 t
134.6 s
25.7 q
17.8 q
22.2 q
26.3 q
29.5 t
125.3 d
133.7 s
25.5 q
17.9 q
28.5 t
5
HL-60 cells (2£10 ) were cultured in 60 mm Petri dishes
and incubated for 12 h. Then cells were harvested, washed
with PBS, resuspended in 200 mL of PBS, and fixed in
8
00 mL of iced 100% ethanol at 2208C. After being left
to stand overnight, the cell pellets were collected by
centrifugation, resuspended in 1 mL of hypotonic buffer
(0.5% Triton X-100 in PBS and 0.5 mg/mL RNase), and
incubated at 378C for 30 min. Then 1 mL of propidium
iodide solution (50 mg/mL) was added, and the mixture was
allowed to stand on ice for 30 min. Fluorescence emitted
from the propidium iodide–DNA complex was quantitated
after excitation of the fluorescent dye by FACScan
cytometry (Becton Dickinson, San Jose, CA).
3
3
3
0
1
2
43.7 d
148.2 s
112.9 t
43.7 d
148.4 s
112.9 t
29.2 d
86.8 s
28.2 t
3
3
3
4
17.8 q
32.8 t
18.0 q
32.9 t
21.1 q
39.3 t
3
3
3
3
5
6
7
8
124.2 d
132.2 s
26.0 q
18.1 q
123.3 d
132.2 s
26.0 q
18.1 q
124.9 d
133.1 s
25.4 q
17.7 q
3
.5. Nitrite assay
The nitrite concentration in the culture medium was
measured as an indicator of NO production, according to
the Griess reaction. One hundred microliters of each
a
Recorded in CDCl
Recorded in CDCl
3
.
b
3
3
–CD OD (5:1).

10102
S. Sang et al. / Tetrahedron 58 (2002) 10095–10102
supernatant was mixed with the same volume of Griess
reagent (1% sulfanilamide in 5% phosphoric acid and
9. Yamaguchi, F.; Saito, M.; Ariga, T.; Yoshimura, Y.;
Nakazawa, H. J. Agric. Food Chem. 2000, 48, 2320–2325.
10. Sang, S. M.; Pan, M. H.; Cheng, X.; Bai, N. S.; Lin-Shiau,
S. Y.; Lin, J. K.; Stark, R. E.; Ghai, G.; Rosen, R.; Ho, C.-T.
Tetrahedron 2001, 57, 9931–9938.
0
.1% naphthylethylenediamine dihydrochloride in water).
Absorbance of the mixture at 550 nm was determined with
an enzyme-linked immunosorbent assay plate reader
(
Labsystems Multiskan RC).
11. Frankel, E. N. Lipid Oxidation. The Oily: Dundee, Scotland,
1
998; pp 129–160.
3
.6. Western blotting
12. El-Feraly, F. S.; Chan, Y. M.; Capiton, G. A.; Doskotch, R. W.;
Fairchild, E. H. J. Org. Chem. 1979, 44, 3952–3955.
3. Christian, O. E.; Henry, G. E.; Jacobs, H.; Mclean, S.;
Total cellular extracts were prepared according to our
previous papers (Pan et al., 2000), separated on 8%
SDS-polyacrylamide minigels, and transferred to immobilin
polyvinylidene difluoride membranes (Millipore). The
membrane was incubated overnight at 48C with 1% BSA
and then incubated with anti-iNOS or anti-a-tubulin
monoclonal antibodies (Transduction Laboratories).
Expression of protein was detected by chemiluminescence
1
1
1
1
1
1
1
Reynolds, W. F. J. Nat. Prod. 2001, 64, 23–25.
4. Abraham, M. H.; Davies, A. G.; Llewellyn, D. R.; Thain, E. M.
Anal. Chim. Acta 1957, 17, 499–503.
5. Rama, R. A. V.; Venkatswamy, G.; Pendse, A. D. Tetrahedron
Lett. 1980, 21, 1975–1978.
6. Ito, C.; Miyamoto, Y.; Nakayama, M.; Kawai, Y.; Rao, K. S.;
Furukawa, H. Chem. Pharm. Bull. 1997, 45, 1403–1413.
7. Yamaguchi, N.; Ariga, T. Jpn Kokai Tokkyo Koho JP
(
ECL, Amersham).
2
000072665 A2, 2000.
8. Yamaguchi, N.; Ariga, T. Jpn Kokai Tokkyo Koho JP
00004468 A2, 2000.
References
. Krishnamurthy, N.; Lewis, Y. S.; Ravindranath, B.
Tetrahedron Lett. 1981, 22, 793–796.
2
9. Yamaguchi, N.; Kataoka, S.; Arika, T.; Murakami, A.;
Koshimizu, K. Jpn Kokai Tokkyo Koho JP 11139965 A2,
1
2
1
999.
. Krishnamurthy, N.; Ravindranath, B.; Row, T. N. G.;
Venkatesan, K. Crystal and molecular structure of isogarcinol.
Tetrahedron Lett. 1982, 23, 2233–2236.
2
2
2
2
0. Saito, M.; Ishikawa, H.; Yamaguchi, N.; Ariga, T.; Kataoka,
S. Jpn Kokai Tokkyo Koho JP 11029465 A2, 1999.
1. Valcic, S.; Muders, A.; Jacobsen, N. E.; Liebler, D. C.;
Timmermann, B. N. Chem. Res. Toxicol. 1999, 12, 382–386.
2. Jovanovic, S. V.; Steenken, S.; Tosic, M.; Marjanovic, B.;
Simic, M. G. J. Am. Chem. Soc. 1994, 116, 4846–4851.
3. Salah, N.; Miller, N. J.; Paganga, G.; Tijburg, L.; Bolwell,
G. P.; Rice-Evans, C. Arch. Biochem. Biophys. 1995, 322,
3
. Bakana, P.; Claeys, M.; Totte, J.; Pieters, L. A. C.; Van Hoof,
L.; Van Den Berghe, D. A.; Vlietink, A. J. J. Ethnopharmacol.
1
987, 21, 75–84.
. Sahu, A.; Das, B.; Chatterjee, A. Phytochemistry 1989, 28,
233–1235.
4
5
1
. Iinuma, M.; Tosa, H.; Tanaka, T.; Kanamaru, S.; Asai, F.;
Kobayashi, Y.; Miyauchi, K.; Shimano, R. Biol. Pharm. Bull.
3
39–346.
2
2
2
2
4. Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Free Rad. Biol.
1996, 19, 311–314.
Med. 1996, 20, 933–956.
6
7
. Yamaguchi, F.; Ariga, T.; Yoshimura, Y.; Nakazawa, H.
J. Agric. Food Chem. 2000, 48, 180–185.
5. Jovanovic, S. V.; Steenken, S.; Hara, Y.; Simic, M. G. J. Chem.
Soc., Perkin Trans. 2 1996, 2497–2504.
6. Prior, R. L.; Cao, G. Proc. Soc. Exp. Biol. Med. 1999, 220,
. Tanaka, T.; Kohno, H.; Shimada, R.; Kagami, S.; Yamaguchi,
F.; Kataoka, S.; Ariga, T.; Murakami, A.; Koshimizu, K.;
Ohigashi, H. Carcinogenesis 2000, 21, 1183–1189.
. Pan, M. H.; Chang, W. L.; Lin-Shiau, S. Y.; Ho, C. T.; Lin,
J. K. J. Agric. Food Chem. 2001, 49, 1464–1474.
2
55–261.
7. Kondo, K.; Kurihara, M.; Miyata, N.; Suzuki, T.; Toyoda, M.
8
Arch. Biochem. Biophys. 1999, 362, 79–86.