PHYTOTHERAPY RESEARCH
Phytother. Res. 17, 485 – 489 (2003)
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1180
John Wiley & Sons, Ltd.
Inhibitory Activity of Plant Extracts on Nitric
Oxide Synthesis in LPS-Activated
Macrophages
Jae-Ha Ryu1*, Hanna Ahn1, Ji Yeon Kim1 and Young-Kyoon Kim2
INHIBITION OF NITRIC OXIDE PRODOCTION BY PCANTS
1
2
College of Pharmacy, Sookmyung Women’s University, Seoul, Korea;
College of Forest Science, Kookmin University, Seoul, Korea
Nitric oxide (NO) produced in large amounts by inducible nitric oxide synthase (iNOS) is known to be
responsible for the vasodilation and hypotension observed in septic shock and inflammation. Inhibitors of
iNOS, thus, may be useful candidates for the treatment of inflammatory diseases accompanied by overproduction of NO. We prepared alcoholic extracts of woody plants and screened the inhibitory activity of NO
production in lipopolysaccharide (LPS)-activated macrophages after the treatment of these extracts. Among
83 kinds of plant extracts, 23 kinds of extracts showed potent inhibitory activity of NO production above 60%
at the concentration of 80 µg/ml. Some of potent extracts showed dose dependent inhibition of NO production
of LPS-activated macrophages at the concentration of 80, 40, 20 µg/ml. Especially, Artemisia iwayomogi,
Machilus thunbergii, Populus davidiana and Populus maximowiczii showed the most potent inhibition
(above 70%) at the concentration of 40 µg/ml. Inhibitory activity of NO production was concentrated to
nonpolar solvent fractions (ethyl ether and/or ethyl acetate soluble fractions) of Artemisia iwayomogi,
Machilus thunbergii and Morus bombycis. These plants are promising candidates for the study of the activityguided purification of active compounds and would be useful for the treatment of inflammatory diseases
and endotoxemia accompanying overproduction of NO. Copyright © 2003 John Wiley & Sons, Ltd.
Keywords: nitric oxide; inhibitor; nitric oxide synthase; plant; macrophage.
INTRODUCTION
L-Arginine-derived nitric oxide (NO) is an intracellular
mediator produced in mammalian cells by two types of
nitric oxide synthase (NOS) (Forstermann et al., 1991).
A constitutive NOS (cNOS) is Ca2+-dependent and
releases small amounts of NO which is required for
physiological functions (Bredt and Snyder, 1990). The
other form of inducible NOS (iNOS) is Ca2+-independent
and induced by lipopolysaccharide (LPS) or some
proinflammatory cytokines such as TNF-α, Il-1β and
IFN-γ (Billiar et al., 1990; Kilbourn and Belloni, 1990;
Stuehr et al., 1991; Iida et al., 1992). NO produced in
large amounts by iNOS and its derivatives, such as
peroxynitrite and nitrogen dioxide, play a role in inflammation and also possibly in the multistage process of
carcinogenesis (Oshima and Bartsch, 1994). NO is also
known to be responsible for the vasodilation and hypotension observed in septic shock (Kilbourn et al., 1990;
Thiemermann and Vane, 1990). Therefore inhibitors of
iNOS may be useful therapeutic agents in septic shock
and inflammaion. Recently, several iNOS inhibitors
were reported from plants such as bisbenzylisoquinoline alkaloids (Kondo et al., 1993), benzoquinones
* Correspondence to: Prof. J.-H. Ryu, College of Pharmacy, Sookmyung
Women’s University, Chungpa-Dong, Yongsan-Ku, Seoul 140-742, Korea.
Tel: 02 710 9568. Fax: 02 714 0745.
E-mail: ryuha@sdic.sookmyung.ac.kr
Copyright © 2003 John Wiley & Sons, Ltd.
(Niwa et al., 1997), sesquiterpene lactones (Park et al.,
1996; Lee et al., 1999), curcuminoids (Brouet and Oshima,
1995; Jang et al., 2001), lignans (Son et al., 2000) and
polyacetylenes (Choi et al., 2000). Most of these compounds showed inhibitory activity of NO production
through the inhibition of iNOS expression.
In order to find new iNOS inhibitors from woody plants,
we have screened inhibitory activity of NO production
by measuring the NO production in LPS-stimulated
RAW 264.7 cells.
MATERIALS AND METHODS
Reagents. The following reagents were used: Dulbecco’s
modified Eagle’s medium (DMEM) (Gibco Laboratories,
Detroit, USA); LPS (Escherichia coli, 0127:B8), bovine
serum albumin, sodium nitrite, N-(1-naphthyl) ethylenediamine and NG-monomethyl-L-arginine (L-NMMA)
(Sigma Chemical Co, St Louis, MO, USA); anti-mouse
i-NOS polyclonal antibody (Transduction Laboratories,
Lexington, KY, USA) and alkaline phosphatase-labeled
goat anti-rabbit antibody (Gibco-BRL, NY, USA).
Plant materials. The plants were collected from PochunGun, the northern part of Kyung-Gi Province, Korea
in November 1997. Vouchers were deposited in the
Laboratory of Forest Chemistry, Department of Forest
Products, Kookmin University. Verification of vouchers or
living plants was performed by Sungsik Kim, Kwangnung
Arboretum, Forest Research Institute, Korea.
Received 17 December 2001
Accepted 22 January 2002
486
J.-H. RYU ET AL.
Extraction and solvent fractionation. The stem barks
of woody plant materials were air-dried, ground and
extracted three times with MeOH at room temperature
and the solvent was evaporated under reduced pressure. The methanolic extracts of plant materials were
dispersed in water and extracted with ethyl ether to
give ether soluble layer. The remaining water layer was
extracted again with EtOAc and n-BuOH sequentially,
to yield EtOAc and BuOH soluble fractions.
Cell culture. The murine macrophage cell line (RAW
264.7) was obtained from the American Type Culture
Collection (Rockville, MD, USA). Cells were cultured
in DMEM containing 10% fetal bovine serum, 2 mM
glutamine, 1 mM pyruvate, penicillin (100 U/ml) and
streptomycin (10 µg/ml). Cells were grown at 37 °C, 5%
CO2 in fully humidified air, and were split twice a week.
RAW 264.7 cells were seeded at 8 × 105 cells/ml in 24
well-plates and were activated by incubation in medium
containing LPS (1 µg/ml) and various concentrations of
test compounds were dissolved in water or DMSO. The
supernatants were collected as sources of secreted NO.
Figure 1. Dose dependent production of NO with various concentrations of LPS (µg/ml) in culture media of RAW 264.7 cells.
The amounts of NO were measured as described in materials
and methods section after incubation with LPS for 20 h.
Nitrite assay. NO released from macrophages was
assessed by the determination of NO−2 concentration in
culture supernatant. Samples (100 µl) of culture media
were incubated with an 150 µl of Griess reagent (1%
sulfanilamide, 0.1% naphthylethylene diamine in 2.5%
phosphoric acid solution) at room temperature for 10 min
in 96-well microplate (Green et al., 1982). Absorbance
at 540 nm was read using an ELISA plate reader. Standard
calibration curves were prepared using sodium nitrite
as standard.
Western blot analysis of i-NOS. The cells were rinsed
with phosphate buffered saline and lysed by boiling
with lysis buffer (1% SDS, 1.0 mM sod. vanadate, 10 mM
Tris, pH 7.4) for 5 min. 30 µg protein of cell lysates was
applied on 8% SDS-polyacrylamide gels (Laemmli, 1970)
and transferred to PVDF membrane by the standard
method. The membrane was blocked with a solution
containing 3% BSA for 1 h. It was then incubated with
anti-mouse i-NOS polyclonal antibody as primary antibody for 2 h and was washed three times with phosphate buffered saline. After incubation with alkaline
phosphatase-labeled goat anti-rabbit antibody for 1 h,
the bands were visualized using nitroblue tetrazolium and
5-bromo-4-chloro-3-indolylphosphate as substrate for
phosphatase (Bio Rad Laboratories, Hercules, CA, USA).
RESULTS ANS DISCUSSION
In order to find new iNOS inhibitors from plants, the
inhibitory activity of NO production was screened by
measuring the NO production in LPS-stimulated RAW
264.7 cells. All the plant samples were dissolved in
dimethyl sulfoxide (DMSO) and diluted with sterile
water for adjusting the concentrations of test samples.
The final concentration of DMSO in culture media was
0.1% and this concentration of DMSO did not show
any effect on the assay systems.
As shown in Fig. 1, the production of NO was
dependent on the concentration of LPS in culture
media. The optimum concentration of LPS was adopted
Copyright © 2003 John Wiley & Sons, Ltd.
Figure 2. Time dependent concentrations of NO produced by
LPS-activated RAW 264.7 cells. After incubation with LPS (1 µg/
ml) for indicated times, the amounts of NO released into culture
media were measured as described in materials and methods
section. The NO of media control group was measured after
20 h incubation without LPS treatment.
as 1 µg/ml that is enough for the induction of iNOS
without cell toxicity. As shown in Fig. 2, the amounts
of NO reached a maximum after incubation with LPS
(1 µg/ml) for 20 h, and decreased slowly with further
incubation. In LPS stimulated RAW 264.7 cell culture
system, the production of NO was increased by the
enzymatic reaction of induced iNOS. The concentration
of NO−2 of LPS-treated group was 30–40 µM, while
those of buffer treated group was less than 8 µM. The
assay samples were added into the culture media of
RAW 264.7 cells during the LPS-activation for 20 h,
and the inhibitory activity of NO production by samples
was calculated by using the followed equation;
Inhibition (%) = 100 × [ODlps − ODsample]/
[ODlps − ODmedia]
The values of OD were measured at 540 nm as
described in materials and methods section for each
treated groups. The inhibition of accumulation of NO
in culture media was 65% by treatment with 0.1 mM
NG-monomethyl-L-arginine (L-NMMA) which is an
inhibitor of NOS through substrate competition (data
not shown).
Phytother. Res. 17, 485 –489 (2003)
487
INHIBITION OF NITRIC OXIDE PRODUCTION BY PLANTS
Table 1. Inhibitory activities of the plant extracts on the LPSactivated NO production in macrophages
Table 1. Continued.
Inhibition (%)a
Botanical name
Botanical name
Abelia mosanensis T. Chung
Abeliophyllum distichum Nakai
Abies holophylla Max.
Abies nephrolepis Max.
Acanthopanax koreanum Nakai
Acanthopanax senticosus (Rupr. et Max.) H.
Acanthopanax sessiliflorus (Rupr. et Max.) S.
Acer tegmentosum Max.
Acer ukurunduensi Trautv. et Meyer
Actinidia arguta Planch.
Alangium platanifoloum var.
macrophyllum (S. et Z.) Wanger.
Auricularia auricula-jade
Berberis koreana Dalibin
Callicarpa japonica Thunb.
Caragana sinica (Buchoz) Rehder
Castanea crenata S. et Z.
Catalpa ovata G. Don
Celtis cordifolia Nakai
Cephalotaxus koreana Nakai
Clerodendron trichotomum Thunb.
Cornus walteri Wanger.
Corylus heterophylla var. thunbergii Bl.
Crataegus pinnatifida Bunge
Elaeagnus multiflora Thunb.
Elaeagnus umbellata Thunb.
Equisetum hyemale L.
Eucommia ulmoides Oliver
Euonymus alatus (Thunb.) Sieb.
Euonymus japonica Thunb.
Euonymus sieboldiana Bl.
Fraxinus rhynchophylla Hance
Fontanesia phyllyreoides Labill.
Ganoderma lucidum Karsten
Gleditsia japonica var koraiensis
(Nak.) Nakai
Hamamelis japonica S. et Z
Hovenia dulcis Thunb.
Juniperus rigida S. et Z.
Kerria japonica (L.) DC
Lagerstroemia india L.
Larix gamelini var. principisruprechtii
(Mayr) Pilger
Larix leptolepis (S. et Z.) Gordon
Lentinula edodes (Berk)Pegler
Lespedeza cyrtobotrya Miq.
Ligustrum obtusifolium S. et Z.
Lonicera japonica Thunb.
Loranthus tanakae Fr. et Sav.
Malus baccata Borkh.
Picea koraiensis Nakai
Picrasma quassioides (D. Don) Benn.
Pourthiaea villosa Decne.
Prunus maximowiczii Rupr.
Prunus mume S. et Z.
Prunus persica (L.) Batsch
Quercus mongolica Fisch.
Rhamnus davurica Pall.
Rhododendron mucronulatum Turcz.
Rhus trichocarpa Miq.
Rhus verniciflua Stokes
Rosa multiflora Thunb.
Salix gracilistyla Miq.
Sambucus williamsii var. coreana Nakai
Schizandra chinensis Baill
Securinega suffruticosa Rehder
Smilax sieboldii Miq.
Copyright © 2003 John Wiley & Sons, Ltd.
Inhibition (%)a
58.7
14.6
28.9
31.7
50.6
47.4
78.7
44.8
67.9
57.0
69.1
<10
25.7
25.2
62.6
57.1
56.8
42.3
<10
45.4
58.3
50.9
62.0
65.1
61.1
50.1
44.6
<10
76.6
36.7
55.7
15.5
48.1
52.2
28.5
<10
50.6
31.8
32.4
35.1
47.9
11.4
49.7
42.9
27.7
36.4
49.5
34.9
25.2
35.3
61.5
28.4
53.3
65.6
45.3
35.8
79.4
60.8
28.6
40.7
39.4
53.7
41.7
39.2
Sorbus commixta Hedl.
Spiraea prunifolia var. simpliciflora Nakai
Styrax japonica S. et Z.
Syringa reticulata var. mandshurica
(Max.) Hara
Symplocos chinensis var. pilosa
(Nak.) Ohwi
Tilia mandshurica Rupr. et Max.
Torreya nucifera Sieb. et Zucc.
Ulmus parvifolia Jacq.
Viburnum sargentii Koehne
Weigela subsessilis L. H. Bailey
Zanthoxylum schinifolium S. et Z.
62.4
36.7
55.3
75.3
66.1
<10
51.8
28.4
34.8
72.6
39.3
a
Final concentrations of samples in culture media were 80
µg/ml.
Table 2. Dose dependent inhibition of NO production by the
plant extracts of plants in LPS-activated macrophages
Botanical name
Inhibition (%)
80
Abies koreana Wilson
Artemisia iwayomogi
Broussonetia kazinoki
Idesia polycarpa
Machilus thunbergii
Morus bombycis
Populus davidiana Dode
Populus maximowiczii
a
a
82.7
94.2
91.9
91.5
97.3
83.5
101.2
91.0
40a
20a
61.5
74.3
68.8
55.9
81.2
54.1
73.6
75.1
17.4
23.1
32.3
12.2
34.3
21.1
45.6
43.1
Final concentration of samples in culture media in µg/ml.
Table 1 showed the inhibitory activity of NO production by plant extracts in LPS-activated macrophages. Of
the 83 kinds of extracts, 23 species showed higher than
60% inhibition of NO production at the concentration of 80 µg /ml of samples in culture media. As shown
in Table 2, some of the potent extracts showed
dose dependent inhibition of NO production of LPSactivated macrophages at the concentration of 80, 40 and
20 µg/ml. Artemisia iwayomogi, Machilus thunbergii,
Populus davidiana and Populus maximowiczii showed
the most potent inhibition above 70% at the concentration of 40 µg/ml. From 6 plants, including species that
showed strong inhibition of NO production, methanol
extracts were prepared in a large scale, and solvent fractions of them were made with increasing the polarity of
solvents. The inhibitory activities of NO production was
concentrated to the certain solvent fractions; to ether
and EtOAc fraction for Artemisia iwayomogi and Morus
bombycis, to ether fraction for Machilus thunbergii, to
EtOAC fraction for Populus davidiana. The viabilities
of RAW 264.7 cells were assessed to be above 85% by
MTT method (Mosmann, 1983) at the sample concentrations for the nitrite assay. The ether soluble fraction
of Rhus verniciflua was toxic against RAW 264.7 cells at
10–50 µg/ml in cell culture media. The inhibitory activity of NO production by medicinal plants may come
from the inhibition of iNOS enzyme activity and/or
expression of nitric oxide synthase. They did not show
Phytother. Res. 17, 485 –489 (2003)
488
J.-H. RYU ET AL.
Table 3. Inhibitory activities of the solvent fractions of plant extracts on the LPS-activated
NO production in macrophages
Botanical name
Solvent
fractions
Inhibition (%)
50
a
30a
10a
Artemisia iwayomogi
Ether
EtOAc
BuOH
107.1
113.5
25.5
106.2
95.1
<10
64.2
57.2
<10
Machilus thunbergii
Ether
EtOAc
BuOH
98.4
51.6
26.5
95.1
34.5
22.2
67.3
29.3
<10
Morus bombycis
Ether
EtOAc
BuOH
105.3
97.8
45.1
93.8
96.4
21.1
67.9
57.2
<10
Populus davidiana Dode
Ether
EtOAc
BuOH
41.6
86.4
73.0
38.3
86.0
58.7
13.7
46.0
32.7
Prunus maximowiczii Rupr.
Ether
EtOAc
BuOH
32.1
26.9
<10
20.2
12.6
<10
12.9
<10
<10
Rhus verniciflua
Ether
EtOAc
BuOH
Toxic
53.2
17.0
Toxic
35.5
<10
Toxic
<10
<10
a
Final concentration of samples in culture media in µg/ml.
Figure 3. Western blot analysis of iNOS in lysates of RAW
264.7 cells (30 µg protein /lane). Cell lysates were prepared as
described in the materials and methods section after 20 h
LPS treatment with samples (30 µg/ml). Lane 1: LPS control,
lane 2: media control, lane 3: ether fraction of Artemisia
iwayomogi, lane 4: EtOAc fraction of Artemisia iwayomogi,
lane 5: ether fraction of Machilus thunbergii, lane 6: EtOAc
fraction of Morus bombycis, lane 7: EtOAC fraction of Populus
davidiana.
any significant inhibitory activities of NO production
when they were added into cell culture media after
induction of iNOS by LPS (data not shown). As shown
in Western blot data of cell lysates (Fig. 3), several
solvent fractions of plants inhibited the expression of
iNOS protein compared with the LPS control group.
Many compounds from medicinal plants have been
known as inhibitors of expression of iNOS in LPS-activated
macrophages. Their structures can be categorized as
sesquiterpene (Park et al., 1996; Lee et al., 1999), flavonoid
(Kim et al., 1999; Kobuchi et al., 1997), polyacetylenes
(Choi et al., 2000) and lignans (Son et al., 2000). The
plants showing inhibitory activity of NO production can
be promising candidates for the activity-guided isolation
of active components having iNOS inhibitory activity,
which may have potential for the treatment of endotoxemia
and inflammation accompanying overproduction of NO.
Further investigation is underway to characterize active
constituents present in the extract of plants.
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