Effects of β-glucosidase hydrolyzed products of harpagide and harpagoside on cyclooxygenase-2 (COX-2) in vitro

Article abstract of DOI:10.1016/j.bmc.2011.06.069

Harpagide (1) and harpagoside (2) are two iridoid glycosides existing in many medicinal plants. Although they are believed to be the main bioactive compounds related to the anti-inflammatory efficacy of these plants, the mechanisms of their anti-inflammatory activities remain unclear. The results of our present study showed that 1 and 2 had no effects on inhibitions of cyclooxygenase (COX)-1/2 enzyme activity, tumor necrosis factor-α (TNF-α) release, and nitric oxide (NO) production in vitro. However, the hydrolyzed products of 1 and 2 with β-glucosidase treatment showed a significant inhibitory effect on COX-2 activity at 2.5-100 μM in a concentration-dependent manner. Our further study revealed that the hydrolyzed 2 product was structurally the same as the hydrolyzed 1 product (H-harpagide (3)). The structure of 3 was 2-(formylmethyl)-2,3,5-trihydroxy-5- methylcyclopentane carbaldehyde, with a backbone similar to prostaglandins and COX-2 inhibitors such as celecoxib. All of them have a pentatomic ring with two adjacent side chains. The result of molecular modeling and docking study showed that 3 could bind to the COX-2 active domain well through hydrophobic and hydrogen-bonding interactions, whereas 1 and 2 could not, implying that the hydrolysis of the glycosidic bond of 1 and 2 is a pre-requisite step for their COX-2 inhibitory activity.

Full text of DOI:10.1016/j.bmc.2011.06.069

Bioorganic & Medicinal Chemistry 19 (2011) 4882–4886
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effects of b-glucosidase hydrolyzed products of harpagide and harpagoside
on cyclooxygenase-2 (COX-2) in vitro
Liuqiang Zhang a, Li Feng a, Qi Jia a, Jinwen Xu b, Rui Wang a, Zhengtao Wang c, Yingchun Wu a, Yiming Li a,
⇑
a School of Pharmacy, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China
b Murad Research Institute for Modernized Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
c Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Harpagide (1) and harpagoside (2) are two iridoid glycosides existing in many medicinal plants. Although
they are believed to be the main bioactive compounds related to the anti-inflammatory efficacy of these
plants, the mechanisms of their anti-inflammatory activities remain unclear. The results of our present
study showed that 1 and 2 had no effects on inhibitions of cyclooxygenase (COX)-1/2 enzyme activity,
Received 23 May 2011
Revised 23 June 2011
Accepted 23 June 2011
Available online 29 June 2011
tumor necrosis factor-
lyzed products of 1 and 2 with b-glucosidase treatment showed a significant inhibitory effect on COX-2
activity at 2.5–100 M in a concentration-dependent manner. Our further study revealed that the hydro-
a (TNF-a) release, and nitric oxide (NO) production in vitro. However, the hydro-
Keywords:
Harpagide
l
lyzed 2 product was structurally the same as the hydrolyzed 1 product (H-harpagide (3)). The structure of
3 was 2-(formylmethyl)-2,3,5-trihydroxy-5-methylcyclopentane carbaldehyde, with a backbone similar
to prostaglandins and COX-2 inhibitors such as celecoxib. All of them have a pentatomic ring with two
adjacent side chains. The result of molecular modeling and docking study showed that 3 could bind to
the COX-2 active domain well through hydrophobic and hydrogen-bonding interactions, whereas 1
and 2 could not, implying that the hydrolysis of the glycosidic bond of 1 and 2 is a pre-requisite step
for their COX-2 inhibitory activity.
Harpagoside
Iridoids
Anti-inflammation
b-Glucosidase
COX-2 inhibitors
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
showed that 1 and 2 had no effect on 5-LOX, iNOS, and LPS-induced
TNF-a
release,8,11 and 2 could even cause a significant increase in
Harpagide (1) and harpagoside (2) are naturally occurring irid-
oids found in many medicinal plants such as Scrophularia ningpoen-
sis,1 Scrophularia buergeriana,2 and Harpagophytum procumbens.3
These medicinal plants have been shown to exhibit a variety of bio-
logical activities and used as pharmaceutical products and dietary
supplements for the treatment of inflammatory ailments, such as
faucitis,4 rheumatoid arthritis5 and osteoarthritis.6 In vitro and
in vivo bioassays have revealed that plants rich in 1 and 2 possess
significant anti-inflammatory activities.7 1 and 2 are believed to be
the main bioactive compounds related to the anti-inflammatory
efficacy of these plants.
the level of COX-2 expression.12
Our previous in vitro assay results (unpublished) showed that 1
had no effect on cyclooxygenase (COX)-1/2 enzymes, tumor necro-
sis factor-a (TNF-a) release, and nitric oxide (NO) production. Our
further experiment indicated that they were not antioxidants,
either.13 Recently, Park et al reported that some iridoid glucosides
(aucubin, catalpol, gentiopicroside, swertiamarin, geniposide, gen-
iposidic acid and loganin) were hydrolyzed by b-glucosidase first
to display their inhibitory effects on COX-1/-2 and TNF-a.
14 In this
study, we attempted to clarify the bioactive form of 1 and 2 by
exploring the structures of compounds derived from b-glucosidase
hydrolysis of 1 and 2 and their anti-inflammatory activities.
The mechanisms of anti-inflammatory efficacies of 1 and 2 are
not fully understood due to controversies in the existing data.
There are studies reporting that 1 and 2 arrested the expression
of COX-2 and its product PGE2;8 suppressed lipopolysaccharide-in-
duced iNOS and COX-2 expression through inhibition of NF-kappa
B activation;9 and inhibited the production of IL-1b, IL-6, and TNF-
2. Results and discussion
2.1. Identification of the structure of the hydrolyzed product of
harpagide (H-harpagide (3))
a
in mouse macrophage cells.10 Several other studies, however,
The structure of 3 was established by spectroscopic data and
references with 1. The ESI-MS spectrum showed that the molecular
weight of 3 was 202 Da. 1H NMR spectrum of 3 contained the
⇑
Corresponding author. Tel.: +86 21 51322191; fax: +86 21 51322193.
E-mail address: ymlius@163.com (Y. Li).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.069

L. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4882–4886
4883
OH
OH
OH
OH
4
4
β-glucosidase
6
5
6
5
CHO
3
7
8
3
7
9
9
O
1
1
8
CHO
HO
10
HO
O Glc
10
H-harpagide
Harpagide
OH
OH
OH
OH
β-glucosidase
CHO
O
+
COOH
CHO
O
O
Glc
HO
O
Cinnamic acid
Harpagoside
H-harpagide
Figure 1. Hydrolysis reaction of 1 and 2 with b-glucosidase.
signals for two aldehyde protons, d 10.16 (1H, s, H-1), d 9.74 (1H, br
s, H-3); a methyl group at d 2.41 (3H, s, H-10) which linked to a
quaternary carbon atom; three protons of ABX system d 5.02 (1H,
d, J = 6.6 Hz, H-6), d 3.09 (1H, dd, J = 21.0, 6.6 Hz, H-7b), d 2.78
(1H, d, J = 21.0 Hz, H-7a); two protons from multiple peaks at d
2.33 (2H, m, H-4). By comparing the spectral data of 1, the struc-
ture of 3 was readily deduced as 2-(formylmethyl)-2,3,5-trihy-
droxy-5-methylcyclopentane carbaldehyde (Fig. 1).
2.2. Effects on cell viability
No significant cytotoxicity on RAW 264.7 macrophages was ob-
served for 1, 2 and 3 at the concentrations observed (2.5–100 lM).
Figure 2. The effect of 3 on COX-1/2. The ovine COX-1 and human recombinant
COX-2 were treated with vehicle (DMSO) or 3 for 10 min. The prostaglandins
formed via COX-1/2 were determined by enzyme immunoassay (EIA). The values
represented mean SEM. The enzymes treated with vehicle (DMSO) were used as
control which represented enzyme 100% initial activity. The statistically significant
2.3. Effect on activities of COX-1/2 enzymes
difference ⁄P <0.05, ⁄⁄P <0.01 compared with control. The data came from
3
The effects of 1, 2, and 3 on ovine COX-1 and human recombi-
nant COX-2 were compared. The COX-1 or COX-2 derived prosta-
noid product was determined via enzyme immunoassay (EIA)
using a broadly specific antibody that bound to all the major pros-
taglandin compounds. The background was detected with inacti-
vated enzymes, and the value was 2.8 0.5 for COX-1 and
2.9 0.2 ng/ml and for COX-2. The enzyme 100% initial activity
was determined by treated the enzyme with vehicle (DMSO), and
the corresponding value was 45.7 6.3 ng/ml for COX-1 and
152.0 25.3 ng/ml for COX-2. Neither 1 nor 2 showed any inhibi-
tion on the two isozymes of COX at the concentrations 2.5–
independent experiments.
2.5. Effects on TNF-a formation and NO production in RAW
264.7 macrophages
Stimulation of RAW 264.7 murine macrophages by LPS resulted
in production of large amounts of inflammatory substances includ-
ing TNF-
264.7 cells treated with LPS showed that they had no inhibitory ef-
fect on the production of TNF- and NO at the concentration range
of 2.5–100 M (data not shown), whereas the two positive drugs
amino guanidine and dexamethasone showed a significant inhibi-
a and NO. Investigation of the effects of 1, 2, and 3 on RAW
100
the product significantly inhibited the activity of COX-2 at concen-
trations 2.5–100 M in a concentration-dependent manner with
the value of IC50 43.3 M, and no inhibition for COX-1 was ob-
l
M. The effect of 3 was also assayed (Fig. 2), showing that
a
l
l
l
tory effect on the production of NO and TNF-a.
served at the same concentration range. Aspirin and NS-398 were
used as positive controls, which significantly inhibited COX-1 or
COX-2 (P <0.01).
2.6. Binding features of 3 with COX-1/2
The calculation results showed that the binding energy of 3
with COX-2 was lower than that of 1 or 2 with COX-2, 3 with the
COX-1, which is consistent with the result of enzyme activity as-
say. The binding interactions of 3 with COX-2 and COX-1 could
be simply described as hydrophobic interactions and hydrogen-
bonding interactions.
2.4. Effects on COX-2 mRNA expression in RAW 264.7
macrophages
The effects of 1, 2, and 3 on COX-2 mRNA expression in RAW
264.7 macrophages were determined by real-time PCR. No signifi-
cant effect on basal mRNA expression of COX-2 was observed for
the three compounds in RAW 264.7 cells at 20 and 100
2 mRNA expression was enhanced significantly by stimulation of
LPS 1 g/ml for 24 h (P <0.01) (Fig. 3). Compound 3 inhibited the
LPS-enhanced COX-2 mRNA expression, with a maximal inhibition
rate of 47.6% at 100 M, but no inhibition was observed for 1 and 2
(data not shown). TPCK 20 M inhibited COX-2 mRNA by 59.5%.
l
M. COX-
2.6.1. Hydrophobic interactions
Figure 4A shows that 3 was surrounded by COX-2 residues
Leu352, Tyr 387, Phe518,Val523, Gly526 and Ala 527 through
hydrophobic interactions, which is consistent with the COX-2
inhibitor SC-558’s. The hydrophobic interaction residues of COX-
1 with 3 (Fig. 4B) were Val349, Leu352, Trp387, Phe518, Gly526,
l
l
l

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L. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4882–4886
Park et al. compared the anti-inflammatory effects of aucubin,
catalpol, geniposide and loganin with their enzyme hydrolyzed-
iridoid products (H-iridoid), and found that the glycosides them-
selves did not show any anti-inflammatory effects, whereas the
hydrolyzed forms were bioactive. Although they presumed that
the structures of H-iridoid products contained a cleavage of mono-
terpene rings, no related details were given.14 In the present study,
we purified 3 and determined its structure for the first time mainly
based on NMR data. A closer look at the structure of 3 revealed an
interesting finding that this structure somewhat looks like PGE2 or
other COX-2 selective inhibitors, all of which have a pentatomic
ring with two adjacent side chains. We think that the structural
similarity to PGE2 makes 3 accessible to the active domain of
COX-2 enzyme. This is probably the reason why 1 showed inhibi-
tion on COX-2 only after it was hydrolyzed and formed the pyra-
noid ring, and why 3 had no effect on TNF or iNOS. This
observation may also help explain the result of Park et al. who
found that genipin had no effect on inhibition of PGE2 formation
in LPS-stimulated RAW264.7 cells while H-geniposide was active
on it. The enzymatic hydrolysis of geniposide produced an active
form of aldehyde acetal, which was then easily transformed to a
dialdehyde structure. However, genipin is stable and cannot be
easily transformed to dialdehyde. H-gentiopicroside and H-swert-
iamarin were not effect on inhibition of PGE2 formation either,
since they cannot form a pentatomic ring; instead, they form a
hexatomic ring with adjacent side chains, making it difficult to ac-
cess the active domain of COX enzyme.
Figure 3. Effects of 1, 2, and
3 on COX-2 mRNA expression in RAW 264.7
macrophages. RAW 264.7 macrophages were pre-treated with 3 for 3 h, and then
stimulated with LPS 1 g/ml for 24 h. Total RNA was extracted and COX-2 mRNA
l
To further investigate the binding feature of 3 to COX-2, molec-
ular modeling and docking were used. The modeling results pro-
vide a satisfactory explanation for the binding mode of 3 with
COX-2. The predicted binding free energy of 3 with COX-2 was
lower than that of 1/2 with COX-2, which is supported by the
stronger hydrophobic interaction and existence of hydrogen bonds
observed in the docking form of 3 to COX-2. All these results and
observations offer a reasonable explanation of why the b-glucosi-
dase hydrolyzed product of 1 is the active form to inhibition
COX-2 effect.
expression was monitored by real-time PCR. The data were normalized with b-actin
and expressed as the fold change in gene expression relative to the untreated cell
group. All values were from
mean SEM versus control ##P <0.01 and LPS treated group ⁄⁄P <0.01. N-p-tosyl-
-phenylalanyl chloromethyl ketone (TPCK) 20 M served as a positive control.
3 independent experiments and expressed as
L
l
Ala527 and Ser530, which is also consistent with COX-1 inhibitor
celecoxib on the whole. The hydrophobic interactions of 3 with
COX-2 and COX-1 were relatively conservative to SC-558 and
celecoxib.
2.6.2. Hydrogen-bonding interactions
3. Experimental
Hydrogen bonding is an important characteristic of the interac-
tions between 3 and COX-2. When bound to COX-2, 3 forms two
hydrogen bonds with COX-2. Figure 4A shows clearly that the oxy-
gen of 3 acts as a donor to form two hydrogen bonds with the hy-
droxyl group OH of Tyr385 and Ser530. Tyr 385 is known to be very
important in keeping the catalysis activation of COX-2.15 There was
no hydrogen bond between 3 and COX-1, indicating that the inhib-
itory effect of 3 on COX-2 was stronger than that on COX-1.
3.1. General experimental procedures
1 and 2 were isolated and purified from the roots of Scrophularia
ningpoensis according to the method reported.1 b-glucosidase (EC
3.2.1.21),
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), arachidonic acid (AA), lipopolysaccharide (LPS,
from Escherichia coli, 011: B4), Aspirin, NS-398, aminoguanidine,
N-p-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), and dexa-
2.7. Discussion
methasone were obtained from Sigma Chemical Co. (St Louis,
MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal
bovine serum (FBS), and other reagents for cell cultures were
obtained from Gibco BRL Life Biotechnologies (Gaithersburg, MD,
USA). The 1H nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker Avance 600 spectrometer (Bruker,
Faellanden, Switzerland).
The above results indicate that 1 and 2 themselves did not show
any anti-inflammatory efficacy in vitro. They are supposed to be in
their active forms by enzymatic hydrolysis of glycosidic bond, lead-
ing to the H-iridoid products. Compound 1 was originally pre-
sumed to produce its aglycone after hydrolysis. However, the
structure of the H-iridoid product of 1 was not just simple removal
of sugar moiety to produce its aglycone form. Since the aldehyde
acetal structure is not stable, the pyranoid ring of the aglycone
was opened to form the dialdehyde structure of a new compound
which exhibited an inhibitory effect on COX-2 and a suppressive
effect on COX-2 mRNA expression. In the enzymatic hydrolysis
process of 2, the unstable aglycone would lose its cinnamic acid
residue when the dialdehyde structure was formed. As a result,
the structure of hydrolyzed products for 1 and 2 was the same.
3.2. Cell line
RAW 264.7 murine cell line was obtained from the Shanghai
Institute of Cell Biology, Chinese Academy of Sciences (Shanghai,
China), and maintained in media recommended by the supplier,
supplemented with 10% FBS (Gibco, Paisley, UK), penicillin
(100 U/ml) and streptomycin (100 lg/ml) in a humidified 5% CO2
atmosphere at 37 °C.

L. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4882–4886
4885
Figure 4. Hydrophobic interactions and hydrogen-bonding interactions of COX-2 with 3 (A) and COX-1 with 3 (B). The 3 atoms and the important residues for inhibitor–
protein hydrophobic interaction are represented by pearlescent marks, respectively. Hydrogen bonds are depicted by green lines.
3.3. Hydrolysis of 2 or 1 with b-glucosidase
J = 6.6 Hz, H-6), 3.09 (1H, dd, J = 21.0, 6.6 Hz, H-7b), 2.78 (1H, d,
J = 21.0 Hz, H-7a), 3.67 (1H, br s, H-9), 2.41 (3H, s, H-10).
b-glucosidase was reported as an acid protease and stable at
50 °C. The reported optimal pH value for the hydrolysis was 4.8.16
Here, the optimal hydrolysis conditions were established as: incu-
bated 1 or 2 with b-glucosidase (5:1 w/w) in the acetic acid solution
(pH 4) at 40 °C for 1 h. Compound 1 was checked and completely
transformed into 3 by the TLC method (CH2Cl2–EtOAc 1:1). At the
end of hydrolysis, the hydrolysis solution was extracted exhaus-
tively with CH2Cl2 for three times. The combined CH2Cl2 solution
was evaporated the solvent in vacuo at room temperature, and the
residue was freeze-dried to get 3. The properties of 3: colorless pow-
der, ESIMS (neg.) m/z 201 [MÀH]À; HRESIMS (neg.) m/z 201.0755
(calcd for C9H13O5, 201.0759.); 1H NMR (CDCl3, 600 MHz), d 10.16
(1H, s, H-1), 9.74 (1H, s, H-3), 2.33 (2H, m, H-4), 5.02 (1H, d,
Meanwhile, the hydrolyzed compounds of 2 were the same as 3
and cinnamic acid, as demonstrated by TLC and 1HNMR.
3.4. Cell viability assay
RAW 264.7 murine macrophages were seeded into 96-well
plates. After co-incubation with the compounds tested or vehicle
for 18 h, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium
bromide (MTT) was added to give a final concentration of 0.5
lg/
ml. With addition of 100 l DMSO to each well, cells were incubated
l
for additional 4 h. The amount of formazan accumulated in the
growth medium was assessed at 570 nm using a microplate reader.

4886
L. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4882–4886
Conditions were considered toxic if the ability of cells to metabolize
MTT to formazan was more than 20% lower than that of the control.
by Dunnett‘s test. Values of P <0.05 were considered statistically
significant.
3.5. Cyclooxygenase-1/2 (COX-1/2) assay
3.10. Molecular modeling and docking
The effects of the compounds on COX-1/2 were determined
with COX inhibitor screening assay kit (Cayman Chemical
Company, Ann Arbor, MI, USA) according to the instruction of the
The crystal structure of COX-2 complexed with the inhibitor
SC-558 (PDB ID:6COX) was downloaded from the RCSB protein
data bank and the PDB ID of COX-1 complexed with the inhibi-
tor celecoxib is 3KK6. The potential of the 3D structures of COX-
2 was assigned with Kollman-united charges encoded in the
molecular modeling software Sybyl 7.3 (Linux-os2x).The initial
structures of 1 and 2 were recovered from PubChem Compound
home (http://www.ncbi.nlm.nih.gov/pccompound). The struc-
tures of 3 and H-harpagoside were built by Chemdraw Ultra
7.0. Partial atomic charges were computed using Sybyl 7.3,
applying Gasteiger-Marsili. For the purpose of tackling the inter-
action mode of the inhibitors with enzyme, the advanced dock-
ing program Autodock 3.0.3 was used to perform the automated
molecular docking. All compounds of the training set were man-
ually docked into the SC-558 (or celecoxib) binding pocket of the
COX-2 (COX-1) enzyme.50 docked structures of the inhibitor
were generated after a reasonable number of evaluations. Finally,
the docked complexes of inhibitor-enzyme were selected accord-
ing to criteria of interacting energy combined with geometrical
matching quality.
kit. Briefly, test compound solution or solvent 20
950 l, heme 10 l, and COX-1 (ovine) or COX-2 (human recombi-
nant) 10 l were incubated for 10 min, and AA 10 mM 10 l was
added to initiate the reaction and incubated for 2 min at 37 °C.
One molar of HCl, 50 l was added to terminate the reaction, and
the samples were then treated with 100 l saturated stannous
ll, reaction buffer
l
l
l
l
l
l
chloride solution for 5 min at room temperature. The activity of
COX-1/2 was determined by quantification of prostanglandins
formed with EIA.
3.6. RNA extraction and reverse transcription real-time
quantitative polymerase chain reaction
The RAW 264.7 murine macrophages were seeded in six-well
plates (2 Â 105 cells per well) for RNA extraction. Cells grown to
confluence were treated with the test compound solution or vehi-
cle solution for 3 h, and then stimulated with LPS 1 lg/ml for 24 h.
Total RNA was extracted using TRIzol reagent (Invitrogen Corpora-
tion, Carlsbad, CA, USA) according to the manufacturer’s instruc-
tion. The cDNA reverse-transcribed from total RNA was subjected
to real-time PCR using fluorescent dye SYBR Green and Thermal
Cycler Dice Real time System TP800 (TaKaRa Bio Inc., Kyoto, Japan).
The reagents used above were from SYBR PrimeScript RT-PCR Kit
(TaKaRa Bio Inc., Kyoto, Japan). The COX-2 primers (Forward 5’
TGTGCGACATACTCAAGC 3’, Reverse 3’ CTGATGCACGTTGTGGAC
5’) were designed according to the corresponding reference
sequences in the NCBI data base. PCR thermocycler conditions
comprised an initial holding at 95 °C for 15 s and subsequently a
two-step PCR programme consisting of 95 °C for 15 s and 60 °C
for 1 min for 40 cycles. Each sample was determined in duplicate.
The relative changes of mRNA between the test and control sam-
ples were quantitated using the 2-DDCt method.17 The data were
normalized with b-actin and expressed as the fold change in gene
expression relative to the untreated cell group.
Acknowledgements
This research program was supported by the National Science
and Technology Major Project of China 2009ZX09103-398; the
Program for Professors of Special Appointment (Eastern Scholar)
in Shanghai Institutions of Higher Learning and the Program for
Shanghai Key Discipline Establishment of TCM Pharmaceutics
(J50302); the ‘‘Xinlin’’ scholars and outstanding team training plan
of SHUTCM and the National Natural Science Foundation of China
(No.81073027).
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.069. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
3.7. Assay of TNF-a formation
RAW264.7 cells (2 Â 105 cells/well) were pretreated with the
test compounds, dexamethasone or vehicle solution for 30 min,
and then co-incubated with LPS (100 ng/ml) for further 6 h. The
References and notes
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3.8. Assay of NO production
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test compounds, aminoguanidine or vehicle solution for 20 min,
and then co-incubated with LPS (1
Hundred microlitre supernatant was incubated with Griess reagent
(50 l 1% sulfanilamide and 50 l 0.1% naphthylethylenediamine
lg/ml) for further 18 h.
l
l
in 2.5% phosphoric acid solution). The absorbance at 570 nm was
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3.9. Statistics
Data were expressed as mean SEM. Statistical analysis of the
data was performed by one way analysis of variances followed