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Summary
Plucheu sagittulis (Lam.) Cabr., a popular medicinal herb grown in South America,
was studied for anti-inflammatory and antioxidant activities. The anti-edema action of
P. sugittulis aqueous extract was assayed in different models of inflammation: 1) the
mouse ear edema test induced by arachidonic acid and croton oil; 2) the rat hind-paw
edema test produced by several inflammatory inductors: carrageenan, dextran.
zymosan, platelet-activating factor (PAF) and arachidonic acid: 3) a subacute model
based on the rat carrageenan air-pouch granuloma test. Blood leukocyte free radical
production was measured by flow cytometry with 2,7-dichlorofluorescin diacetate
(DCFH-DA) in vivo, in rats with induced air-pouch granuloma, and in a model in
vitro, stimulating leukocytes with hydrogen peroxide. The aqueous extract of P.
sugittulis showed a marked anti-inflammatory effect in both ear edema tests, dextran
and carrageenan hind-paw edemas and carrageenan air-pouch model. It also had a
potent antioxidant activity in blood leukocytes, both in vivo and in vitro. Our results
correlate the reduction of free radical production with the anti-inflammatory effect of
this plant.
Plucheu sugittulis is widely used in the traditional medicine of South America for digestive
diseases (I). This plant has shown anti-inflammatory activity in a previous plant screening study (2).
Other studies have been reported on the anti-inflammatory activity of different Plucheu sp., as P.
Lunceokm (3) or P. Mica (4).
Several anti-inflammatory drugs have recently shown an antioxidant mechanism as part of their
activity, such as nimesulide (5) and aminosalicylates (6) among others. Thus, it is interesting to study
whether products of natural origin, such plant extracts used in Traditional Medicine, also have an
antioxidant mechanism.
*Corresponding author: Esther Marin
2034
Anti-inflammatory
Reactive oxygen species (ROS) participate in normal cell function as well in pathology. ROS
are produced from different enzymes (e.g. NADPH oxidase, xanthine oxidase, lipoxygenases,
prostaglandin synthase, myeloperoxidase, cytochrome oxidase) and consumed by antioxidant
enzymes (e.g. superoxide dismutase. catalase, glutathione peroxidase) and antioxidant substances
(e.g. vitamins C and E, endogenous chelators, glutathione) reaching to an equilibrium in the
antioxidant-prooxidant balance. Synthesis of these molecules occurs in cells in response to normal
physiological stimuli, but when their concentrations increase above a certain level they can be
harmful. In many pathological conditions, as in inflammatory disease, this equilibrium is broken
producing an increase of ROS, which destroys the antigen that produces the inflammatory process.
But this overproduction leads to tissue injury, by damaging macromolecules and lipid peroxidation of
membrane. In addition, ROS could act as second messengers, activating production of other
mediators involved in the inflammatory process (7-l 1).
In this paper, the anti-inflammatory activity of P. sugittulis is studied in several models, in rats
and mice, in vivo and in vitro, and it is proposed that this activity is, in part, caused by an antioxidant
mechanism.
Methods
Plant collection and extraction
Whole plants of Pluchea sugittulis (Lam.) Cabrera were collected and authenticated in Salta
(Argentine) by Prof. Dr. L. Novara. Aqueous extracts were obtained by decoction of 25 g of ground
dried plant material in 750 ml of water. The decoction was filtered and lyophilized.
Mouse ear edema tests
Of 20-25 g.
Arachidonic acid-induced ear edema. The topical anti-inflammatory effect of the aqueous extract (100
pg/ear) or indomethacin (0.2 pmol/ear) applied 30 min before arachidonic acid (2 mg/ear) were
studied by measuring ear weight one hour after induction of inflammation (12).
Croton oil-induced ear edema. The topical anti-inflammatory activity of the aqueous extract (100
pg/ear) or indomethacin (0.2 pmol/ear) applied along with croton oil (70 pg/ear) were studied by
measuring ear weight six hours after induction of inflammation (13).
Rat hind paw edema tests
Phrcheusagiffulis
203.5
Zvmosan test. The activity of aqueous extract (100 mg/kg i.p.) or methysergide (1 mg/kg i.p.)
against zymosan induced inflammation in the left hind foot (0.1 ml of 1% suspension of zymosan A
in saline) were recorded plethysmographically for 90 min (16).
Platelet-activating factor (PAF) test. The effect of aqueous extract (100 mgikg Lp.) or
dexamethasone (1 mg/kg i.p.) against PAF induced inflammation in the left hind foot (0.1 ml of 10
pg/ml of PAF in saline-with 0.25% bovine serum albumin) were recorded plethysmographically for
90 min (17).
Arachidonic acid test. The activity of aqueous extract (100 mg/kg i.p.) or dexamethasone (1 mg/kg
i.p.) against arachidonic acid foot induced inflammation (0.1 ml of 0.5% arachidonic acid in 0.2 M
buffer solution of sodium carbonate 0.2M) were recorded pletysmographically for 90 min (18).
Carrageenan air-pouch test.
Animals used: Male Sprague-Dawley rats of 130-160 g (Letica, Barcelona).
The anti-inflammatory effect of the extract or phenylbutazone. both 100 mg /kg S.C. in the six day
carrageenan air-pouch edema was studied by measuring exudate volume, granulomatous tissue weight
and the number of cells present (Counter Coulter Multisizer II cytometer, N.J., USA) (19).
Antioxidant activity
Animals used: Male Sprague-Dawley rats of 130-160 g (Letica, Barcelona).
Leukocvte nurification: Blood was collected from rats in heparinized polypropylene tubes. For
separation of leukocytes, 1.5 ml of 6% (w/v) dextran 500 kD in saline and 0.3 ml of NaCl 1.6M
were added to 5 ml of blood. When hemaglutination occurred (ca. 30 min), the upper part of the
sample was removed. This part, rich in leukocytes, called buffy coat, was lysed hyposmotically with
a solution of NaC10.2 % for 20 s. Viability of purified leukocytes was more than 95 %.
Leukocvte antioxidant activi&: 2 ,7-Dichlorofluorescin diacetate (DCFH-DA) is a fluorescence
probe to detect hydrogen peroxide as a measure of reactive oxygen species by flow cytometry (20).
After uptake of DCFH-DA by the cells, this lipophilic compound is deacetyled to dichlorofluorescin
(DCFH) by a cell esterase. DCFH is trapped inside because it is more polar than diacetate and cannot
permeate the membrane. If oxyradicals are present in the cells, they may oxidize the DCFH to
fluorescent dichlorofluorescein (DCF).
Leukocytes were incubated for 10 min at 37 OC with DCFH-DA (Serva, Heidelberg, Germany) 10
uM in the presence of I mM of sodium azide. An Epics Elite flow cytometer (Coulter, N.J.,
U.S.A.) was used for leukocyte population cell identification (forward scatter and side scatter
measurements) and for cell fluorescence intensity analysis.
Antioxidant effect in vivo. The effect of the extract or phenylbutazone, both 100 mg/kg S.C. were
studied in leukocytes from six day carrageenan air-pouch edema rats by measuring reactive oxygen
species by flow cytometry.
Antioxidant effect in vitro. The antioxidant activity of the extract or quercetin were studied in rat
blood leukocytes. After preincubation with DCFH-DA (10 min), cells were incubated with the
product under study (5 min) and finally stimulated with H20z 100 FM (5 min). Dose-response curves
2036
110
110
100
100
90
90 -
80-
60
60 -
50
50-
40
40-
30
L
if
60
70
70:
m-
20
20
10
10
0 L
0
CTR
IND
EXT
CTR
TREATMENT
EXT
IND
TREATMENT
(a)
(b)
Fig. 1
Topical anti-inflammatory activity of Plucheu sagittulis on ear mouse edema tests. The inflammatory
agents were (a) arachidonic acid and (b) croton oil. CTR: control animals; IND: animals treated with
0.2 pmohear of indomethacin, and EXT: animals treated with Pkheu sugittulis aqueous extract 100
pg/ear. (Mean + SEM, N= 10, * ~~0.05).
,
0
OT
TIME Ihl
I
5
TIME fh)
(4
@I
Fig. 2
Time course of anti-inflammatory activity of Plucheu sagiftalis on rat hind-paw edema tests. The
inflammatory agents were (a) carrageenan and (b) dextran. Products tested: sodium naproxen (+) 5
mg/kg, methysergide (m) 1 mg/kg, and Plucheu sugiftulis aqueous extracts from aerial parts (A),
underground parts (A) and the whole plant (0) 100 mg/kg (Mean f SEM, N=6, ~~0.05).
2037
were plotted at pg/ml range for P. sugittufis aqueous extract and quercetin.
Statistical calculations
The results were analyzed by means of analysis of variance and Scheffe test.
ReSUltS
Mice ear edema tests
Arachidonic acid-induced ear edema. Application of P. sugittulis aqueous extract (100 pg/ear) had an
inhibitory effect on inflammation of 44.3 % (Figure la).
Croton oil-induced ear edema. P. sugittulis aqueous extract at 100 Kg/ear produced a 53.1%
inhibition of the edema (Figure lb).
Rat hind paw edema tests
Carrageenan test. lnflammation induced by carrageenan in the rat paw was inhibited by more than
50 % for 5 h by aqueous extracts of aerial parts. underground parts and the whole plant of P.
sugitmlis, following a single dose of 100 mg/kg i.p.(Figure 2a).
Dextran test. An inhibition of inflammation of 63.3% was produced by a single dose of 100 mg/kg
P. sagittufis aqueous extract i.p. The time course of the anti-edema action is represented in Figure
2b.
Om.
In zymosan, PAF and arachidonic acid rat hind-paw edemas, P. sugittulis
aqueous extract 100 mg/kg i.p. did not show any significant anti-inflammatory effect.
Carrageenan air-pouch test
Measurement of the inflammation. The edema was strongly inhibited by P. sugittulis aqueous extract
100 mg/kg s.c.: the exudate volume reduction was 66.1% while decrease in the weight of the
granulomatous tissue was 73.9% (Table I).
Reactive oxygen species evaluation
(DCFH-DA)
by flow cytometry
with 2,7-dichlorofluorescin
diacetate
Test in vivo. A significant inhibition of the free radical formation was observed: 84.3% in
lymphocytes and 89.0% in polymorphonuclear blood leukocytes from the same rats of the previous
experiment (Table II).
Test in vitro. Oxidative stress of rat blood leukocytes stimulated with 100 yM hydrogen peroxide was
inhibited in a dosedependent manner by P. sugittulis aqueous extract at a ugg/mlrange (Figure 3).
2038
TABLE I
PHB
EXT
PHB
EXT
WEIGHT (g)
10.50 f 0.4
2.46 f 0.58
3.56 &-0.86
76.6
66.1
VOLUME (ml)
17.6 + 1.5
5.2 + 1.5
70.5*
73.9*
CELLS x lO/ml
28.6 f 5.6
29.3 +_3.5
37.8 f 6.8
NS
NS
Inflammation is represented as increment in weight of the wet granulomatous tissue and volume of
the inflammatory exudate. Exudate cell number is also shown. PHB: phenylbutazone 100 mg/kg S.C.
daily, EXT: Pfuchea sagiztafis aqueous extract 100 mg/kg S.C. daily (Mean f SEM. N=8,
* p<O.ool).
TABLE II
Effect of Plwhea sagittalison reactive oxygen species production in rat carrageenan air-pouch test.
INHIBITION ( %)
LEUKOCYTE
POPULATION
PHB
EXT
PHB
EXT
LYMPHOCYTES
8.91 f 2.94
5.52 f 1.77
1.40 + 0.50
38.1
84.3.
PMN
27.3 + 8.1
19.6 f 6.5
3.0 + 1.4
28.3
89.0.
PMN: polymorphonuclear cells. PHB: phenylbutazone 100 mg/kg S.C. daily; EXT: Pfuchea
sagittalisaqueous extract 100 mg/kg S.C.daily (Mean + SEM, N=8, * p<O.OOl).
-1
Fig. 3
Dose-response curves of the inhibitory action of Pluchea sagittalis aqueous extract (0) and quercetin
(*) in rat polymorphonuclear cells stimulated with 100 yM hydrogen peroxide (Mean + SEM, 4
experiments, N =3).
2039
Discussion
Plucheu sagitfulis inhibited several inflammatory processes induced by different inflammatory
agents in rats and mice. Carrageenan rat hind-paw test was inhibited by aerial, underground and
whole plant extracts, indicating that the activity was not concentrated in a particular part of the plant
(Fig 2a). Carrageenan-induced edema formation in rats is a general test for anti-inflammatory agents
and it was recently shown the importance of ROS over arachidonic acid metabolites in the
maintenance of carrageenan inflammation (21). ROS are produced during phagocytosis in dextran
induced inflammation as well as vasoactive amines (22, 23). In mouse ear topical inflammation ROS
production is important since antioxidant substances are active (24, 25) and latest data indicate that
eicosanoids are not so important (25, 26). The sixday air-pouch test is a general model of semiacute
inflammation that shows the formation of granulomatous tissue and leukocyte extravasation similar to
human rheumatoid arthritis. In this model of inflammation, ROS production is an important factor
for inflammation development (27). The extract does not inhibit reduction in leukocyte extravasation
as no reduction is observed in exudate cell number (Table II). It is important to point that ROS have
an important role in inflammation. not only as tissue damaging agents, since free radicals activate
arachidonic acid metabolism (especially the inducible cyclooxygenase COX-2), pro-inflammatory
cytokines (i.e. interleukins IL-1 and IL-6. and tumor necrosis factor), heat shock proteins and the
apoptotic process (7-11, 28, 29)
2040
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