Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2011, Article ID 478246, 8 pages
AnalgesicEffectsand theMechanismsof Anti-Inflammation of
Hispolon in Mice
1Institute of Chinese Pharmaceutical Sciences, Taichung 40402, Taiwan
2School of Pharmacy, College of Pharmacy, China Medical University, Taichung 404, Taiwan
3Department of Pesticide Toxicology, Taiwan Agricultural Chemicals and Toxic substances Research institute,
Wu Feng, Taichung 433, Taiwan
Correspondence should be addressed to Guan-Jhong Huang, firstname.lastname@example.org
Received 29 October 2008; Accepted 3 March 2009
Copyright © 2011 Heng-Yuan Chang et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Hispolon, an active ingredient in the fungi Phellinus linteus was evaluated with analgesic and anti-inflammatory effects. Treatment
of male ICR mice with hispolon (10 and 20mg/kg) significantly inhibited the numbers of acetic acid-induced writhing response.
Also, our result showed that hispolon (20mg/kg) significantly inhibited the formalin-induced pain in the later phase (P<.01).
In the anti-inflammatory test, hispolon (20mg/kg) decreased the paw edema at the fourth and fifth hour after λ-carrageenin
(Carr) administration, and increased the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione
reductase (GRx) in the liver tissue. We also demonstrated that hispolon significantly attenuated the malondialdehyde (MDA) level
in the edema paw at the fifth hour after Carr injection. Hispolon (10 and 20mg/kg) decreased the nitric oxide (NO) levels on
both the edema paw and serum level at the fifth hour after Carr injection. Also, hispolon (10 and 20mg/kg) diminished the serum
level of MDA in the edema paw by increasing the activities of SOD, GPx and GRx in the liver. It probably exerts anti-inflammatory
effects through the suppression of TNF-α and NO.
In Asia, macrofungi are commonly given as a nutritional
supplement to patients with a variety of diseases . Several
different species of Phellinus are believed to have anticancer
as well as antioxidant properties. Phellinus linteus (PL) has
been demonstrated to exhibit anti-tumor and antioxidant
properties . The methanolic extract of the basidiocarps of
PL exhibits an antioxidative effect . Studies have indicated
that PL could protect primary cultured rat hepatocytes
against hepatotoxins , and P. rimosus (Berk) Pilat seems
to exhibit antioxidant and antihepatotoxic activities .
There is also a report indicating that n-BuOH subfraction
of PL exhibited anti-inflammatory activity . Hispolon,
an active ingredient in the fungi P. igniarius  has
been reported to induce apoptosis in human epidermoid
KB cells . Hispolon has also been shown to inhibit
the chemiluminescence response of human mononuclear
cells and suppress mitogen-induced proliferation of spleen
lymphocytes in mice . In our previous study, we found
activities in a series of in vitro tests . PM also showed
hepatoprotective and antioxidant effects in Sprague-Dawley
rat with carbon-tetrachloride-induced liver damage .
However, little information is available on the analgesic and
anti-inflammatory effects of hispolon.
Some researches demonstrate that inflammatory effect
induced by Carr could be associated with free radical. Free
radical, prostaglandin and nitric oxide (NO) were released
when Carr was administered for 1–6h . The edema
effect was raised to maximum at the third hour  and its
MDA production was due to free-radical attack on plasma
membrane . Thus, inflammatory effect would result in
the accumulation of MDA. Therefore, in this article we
examined the analgesic effects of hispolon on nociception
induced by acetic acid and formalin. We also evaluated the
2Evidence-Based Complementary and Alternative Medicine
Figure 1: Chemical structure of hispolon.
NO and TNF-α in either paw edema or serum. Also, the
activities of SOD, GPx and GRx in the liver at the fifth
hour after Carr injection were investigated to understand the
relationship between the anti-inflammatory mechanism of
hispolon and antioxidant enzymes.
2.1. Chemicals. Hispolon (Figure 1) was synthesized as
described and its purity was established on the basis of the
spectra (1H,13C NMR and mass) data . Acetic acid
was purchased from Merck (Darmstadt, Germany). Carr
and indomethacin were obtained from Sigma (St Louis,
MO, USA). Formalin was purchased from Nihon Shiyaku
Industries (Japan). TNF-α was purchased from Biosource
International Inc. (Camarillo, CA, USA).
2.2. Animals. Six- to eight-week-old male ICR mice were
obtained from the BioLASCO Taiwan Co., Ltd. The animals
were kept in plexiglass cages at a constant temperature of
22±1◦C, relative humidity 55±5% with 12h dark–light
cycle for at least 2 weeks before the experiment. They were
Use of Laboratory Animals. The placebo groups were given
0.1ml/10g saline intraperitoneally using a bent blunted
27-gauge needle connected to a 1mL syringe. All tests
were conducted under the guidelines of the International
Association for the Study of Pain .
After a 2-week adaptation period, male ICR mice (18–
25g) were randomly assigned to five groups (n=8) in acetic-
acid-induced writhing and formalin-induced licking groups.
These include a normal (received acetic acid or formalin),
a positive control (acetic acid or formalin+indomethacin
(Indo)) and hispolon administered groups (acetic acid
or formalin+hispolon).In the Carr-induced edema
experiment, there were six groups (n=8) of the animals
in the study. The control group received normal saline.
The other five groups included a Carr-treated, a positive
control (Carr+Indo) and hispolon-administered groups
2.3. Acetic-Acid-Induced Writhing Response. The test was
performed as described by Fontenele et al. . Nocicep-
tion was induced by an intraperitoneal (i.p.) injection of
0.1mL/10g acetic acid solution (10mL/kg). Positive control
animals were pretreated with Indo (10mg/kg, i.p.) 25min
before acetic acid. Each hispolon administered group was
pretreated with 5mg/kg, 10mg/kg or 20mg/kg i.p. 25min
before acetic acid. Five minutes after the i.p. injection of
acetic acid, the number of writhing and stretching was
2.4. Formalin Test. The antinociceptive activity of the drugs
was determined using the formalin test described by Dubuis-
son and Dennis . Twenty microliters of 5% formalin was
injected into the dorsal surface of the right hind paw of mice
30min after i.p. administration of the hispolon (5, 10 and
injection of formalin, and the amount of time spent licking
the injected hind paw was recorded. The first 5min post
formalin injection is referred to as the early phase and the
time spent licking or biting the injured paw (pain behavior)
was measured with a stop watch. The activity was recorded
in 5min intervals.
2.5. λ-Carrageenin-Induced Edema. Carr-induced hind paw
edema model was used for determination of anti-inflam-
matory activity . Animals were i.p. treated with the his-
polon (5, 10 and 20mg/kg), Indo or normal control, 30min
prior to injection of 1% Carr (50μl) in the plantar side of
right hind paws of the mice. Paw volume was measured
immediately after Carr injection and at 1, 2, 3, 4 and 5h
intervals after the administration of the edem-atogenic agent
using a plethysmometer (model 7159, Ugo Basile, Varese,
Italy). The degree of swelling induced was evaluated by the
ratio a/b, where a is the volume of the right hind paw
after Carr treatment, and b is the volume of the right hind
paw before Carr treatment. Indo was used as a positive
control. After 5h, the animals were sacrificed, the Carr-
induced edema feet were dissected and stored at −80◦C.
Blood samples were withdrawn and kept at −80◦C.
2.5.1. Total Protein Assay. The protein concentration of the
sample was determined by the Bradford dye-binding assay
(Bio-Rad, Hercules, CA).
2.5.2. MDA Assay. MDA from Carr-induced edema foot
was evaluated by the thiobarbituric acid reacting substance
(TRARS) method . Briefly, MDA reacted with thiobarbi-
turic acid in the acidic high temperature and formed a red-
complex TBARS. The absorbance of TBARS was determined
2.5.3. Determination of NO. NO determinations were car-
ried out in 100μL aliquots of sample mixed with 100μL
of the Griess reagent . Nitrite was quantified by using
sodium nitrate as a standard curve.
2.5.4. Measurement of Serum TNF-α by ELISA. Serum levels
of TNF-α were determined using a commercially available
enzyme linked immunosorbent assay (ELISA) kit according
Evidence-Based Complementary and Alternative Medicine3
to the manufacturer’s instruction. TNF-α was determined
from a standard curve for the combination of these
cytokines. The concentrations were expressed as pg/mL .
2.5.5. Antioxidant Enzymes Activity Measurements. Liver
tissue homogenates were collected for the estimation of
SOD , GPx  and GRx enzyme  to detect the
antioxidant activities of hispolon.
2.5.6. Histological Examination. For histological examina-
tion, biopsies of paws were taken 5h following the inter-
planetary injection of Carr. The tissue slices were fixed in
(1.85% formaldehyde, 1% acetic acid) for 1 week at room
temperature, dehydrated by graded ethanol and embedded
in Paraffin (Sherwood Medical). Sections (thickness 5μm)
were deparaffinized with xylene and stained with H & E
stain. All samples were observed and photographed with
BH2 Olympus microscopy. Every three to five tissue slices
were randomly chosen from Carr-, Indo- and hispolon-
treated (20mg/kg) groups. The numbers of neutrophils were
counted in each scope (400x) and thereafter obtain their
average count from five scopes of every tissue slice.
Statistical evaluation was carried out by one-way analysis of
variance (ANOVA followed by Scheffe’s multiple range test).
Statistical significance is expressed as∗P< .05,∗∗P< .01,
3.1. Effects of Hispolon on Acetic-Acid-Induced Writhing
Re-sponse. The cumulative amount of abdominal stretch-
ing correlated with the level of acetic-acid-induced pain
(Figure 2). Hispolon treatment (10mg/kg) significantly
inhibited the number of writhing in comparison with
the normal controls (P< .05). Hispolon (20mg/kg) fur-
ther inhibited the number of writhing (P < .001), and it
demonstrates more inhibition than that produced by Indo
3.2. Formalin Test. Hispolon (20mg/kg) significantly (P<
.01) inhibited formalin-induced pain in the late phase
(Figure 3); however, it did not show any inhibition in
the early phase. The positive control Indo (10mg/kg) also
significantly (P< .001) inhibited the formalin-induced pain
in the late phase.
3.3. Effects of Hispolon on λ-Carrageenan-Induced Mice Paw
Edema. As shown in Figure 4, Carr induced paw edema.
Hispolon (20mg/kg) significantly inhibited (P< .001) the
treatment. Indo (10mg/kg) significantly decreased the Carr-
induced paw edema after 4 and 5h of treatment (P< .001).
3.3.1. Effects of Hispolon on MDA Level Measurements. MDA
level increased significantly in the edema paw at the 5h after
Figure 2: Analagesic effects of the hispolon and indomethacin
(Indo) on acetic-acid-induced writhing response in mice. Each
value represents as mean±SEM.
by Scheffe’s multiple range test).
∗P< .05 and
∗∗∗P< .001 as
Licking times (sec.)
Figure 3: Effects of the hispolon and indomethacin (Indo) on
the early phase and late phase in formalin test in mice. Each
value represents as mean±SEM.
by Scheffe’s multiple range test).
∗∗P< .01 and
∗∗∗P< .001 as
(P< .001 or P< .001), as well as 10mg/kg Indo (Figure 5).
3.3.2. Effects of Hispolon on NO Measurement. In Figure 6,
the NO level increased significantly in the edema paw and
serum at the 5h after Carr injection (P< .001). Hispolon (10
and 20mg/kg) significantly decreased the serum NO level
(P< .05 or P< .01). The inhibitory potency was similar to
that of Indo (10mg/kg) at 5h after induction.
3.3.3. Effects of Hispolon on TNF-α Measurement. TNF-α
level increased significantly in serum at the 5h after Carr
4Evidence-Based Complementary and Alternative Medicine
Carra + Indo
Carra + hispolon 5mg/kg
Carra + hispolon 10mg/kg
Carra + hispolon 20mg/kg
Changes of edema volume (mL)
Figure 4: Effects of the hispolon and indomethacin (Indo) on
hind paw edema induced by λ-carrageenan (Carr) in mice. Each
value represents as mean±SEM.∗∗∗P< .001 as compared with the
Carr group (one-way ANOVA followed by Scheffe’s multiple range
5 1020 (mg/kg)
Tissue MDA concentration
Figure 5: Effects of the hispolon and indomethacin (Indo) on the
tissue MDA concentration of foot in mice. Each value represents
as mean±SEM.###P< .001 as compared with the control group.
∗∗P< .01 and∗∗∗P< .001 as compared with the Carr group (one-
way ANOVA followed by Scheffe’s multiple range test).
injection (P< .001). However, hispolon (10 and 20mg/kg)
decreased the TNF-α level in serum at the 5h after Carr
injection (P< .05 or P< .01), as well as 10mg/kg Indo
3.3.4. Effects of Hispolon on Activities of Antioxidant Enzymes.
At the fifth hour following the intrapaw injection of
5 1020 (mg/kg)
Figure 6: Effects of the hispolon and indomethacin (Indo) on
carrageenan (Carr)-induced NO concentration of edema paw and
serum at fifth hour in mice. Each value represents as mean±SEM.
###P< .001ascomparedwiththecontrolgroup.∗P< .05,∗∗P< .01
ANOVA followed by Scheffe’s multiple range test).
∗∗∗P< .001 as compared with the Carr group (one-way
5 10 20 (mg/kg)
Serum TNF-α (pg/mL)
Figure 7: Effects of the hispolon and indomethacin (Indo) on
carrageenan (Carr)-induced NO concentration of serum at fifth
hour in mice. Each value represents as mean±S.E.M.###P< .001
as compared with the control group.
compared with the Carr group (one-way ANOVA followed by
Scheffe’s multiple range test).
∗P< .05 and
∗∗P< .01 as
Carr, liver tissues were also analyzed for the biochemical
parameters such as SOD, GPx and GRx activities (Table 1).
SOD activity in liver tissue was decreased significantly by
after treated with 20mg/kg hispolon and 10mg/kg Indo
(P< .01) (Table 1). Carr administration markedly decreased
GPx and GRx activities in the livers. GPx and GRx activities
Evidence-Based Complementary and Alternative Medicine5
Table 1: Effects of hispolon and indomethacin (Indo) on the liver SOD, GPx and GRx activities in mice.
Each value represents as mean SEM.∗P< .05 and∗∗P< .01 as compared with the Carr (λ-carrageenan) group (one-way ANOVA followed by Scheffe’s
multiple range test).
SOD (U/mg protein)
GPx (U/mg protein)
GRx (U/mg protein)
of the livers were increased significantly by hispolon (10 and
20mg/kg), as well as Indo (10mg/kg) (Table 1).
3.3.5. Histological Examination. Paw biopsies of control
tissue. The infiltrates accumulated between collagen fibers
and into intercellular spaces. Paw biopsies of animals treated
with hispolon (20mg/kg) showed a reduction in Carr-
induced inflammatory response. Actually inflammatory cells
were reduced in number and confined near to the vas-
cular areas. Intercellular spaces did not show any cellular
infiltrations. Collagen fibers were regular in shape and
showed a reduction of intercellular spaces. Moreover, the
hypoderm connective tissue was not damaged (Figure 8).
Neutrophils were increased with Carr treatment (P< .001).
Indo and hispolon (20mg/kg) could significantly decrease
the neutrophils numbers as compared to the Carr-treated
group (P< .01) (Figure 8(e)).
We have evaluated the putative analgesic and anti-
inflammatory activities of hispolon to clarify the pain and
methods were employed with the objective of identifying
possible peripheral and central effects of the test substances.
The acetic-acid writhing test is normally used to study the
peripheral analgesic effects of drugs. Although this test is
non-specific (e.g. anticholinergic, antihistaminic and other
agents also show activity in the test), it is widely used
for analgesic screening . In our study, we found that
hispolon (10 and 20mg/kg) exhibited antinociceptive effect
in acetic-acid-induced writhing response (Figure 2). This
effect may be due to inhibition of the synthesis of the
arachidonic acid metabolites .
The in vivo model of pain, formalin-induced paw pain
has been well established as a valid model for analgesic
and different analgesics may act differently in the early and
late phases of this test. Therefore, the test can be used to
clarify the possible mechanism of an antinociceptive effect
of a proposed analgesic . Centrally acting drugs such
as opioids inhibit both phases equally , but peripherally
acting drugs such as aspirin, Indo and dexamethasone only
inhibit the late phase. The inhibitory effect of hispolon on
the nociceptive response in the late phase of the formalin
test suggested that the anti-nociceptive effect of the hispolon
could be due to its peripheral action (Figure 3).
The Carr test is highly sensitive to non-steroidal anti-
inflammatory drugs, and has long been accepted as a useful
phlogistic tool for investigating new drug therapies . The
degree of swelling of the Carr-injected paws was maximal
3h after injection. Statistical analysis revealed that hispolon
and Indo significantly inhibited the development of edema
4h after treatment (P< .001) (Figure 4). They both showed
anti-inflammatory effects in Carr-induced mice edema paw.
It is well known that the third phase of the edema-induced
by Carr, in which the edema reaches its highest volume, is
characterized by the presence of prostaglandins and other
compounds of slow reaction . It was found that the
injection of Carr into the rat paw induces the liberation
of bradykinin, which later induces the biosynthesis of
prostaglandin and other autacoids, which are responsible for
the formation of the inflammatory exudates. In addition,
the classification of antinociceptive drugs is usually based
on their mechanism of action either on the central nervous
system or on the peripheral nervous system .
In the studies of mechanism on the inflammation,
l-arginine-NO pathway has been proposed to play an
important role in the Carr-induced inflammatory response
. Our present results also confirm that Carr-induced
paw edema model results in the production of NO. The
expression of the inducible isoform of NO synthase has been
proposed as an important mediator of inflammation .
In our study, the level of NO was decreased significantly
by treatment with 10 and 20mg/kg hispolon. We suggest
the mechanism of anti-inflammatory of hispolon may be
through the l-arginine-NO pathway since hispolon signifi-
cantly inhibit the NO production (Figure 6).
TNF-α is a major mediator in inflammatory responses,
inducing innate immune responses by activating T cells and
macrophages, and stimulating secretion of other inflamma-
tory cytokines . Also, TNF-α is a mediator of Carr-
induced inflammatory incapacitation, and is able to induce
the further release of kinins and leukotrienes, which is
suggested to have an important role in the maintenance
of long-lasting nociceptive response . In this study, we
found hispolon decreased the TNF-α level in serum after
Carr injection (Figure 7).
The Carr-induced inflammatory response has been
linked to neutrophils infiltration and the production of
neutrophils-derived free radicals, such as hydrogen peroxide,
6Evidence-Based Complementary and Alternative Medicine
(Carr) stained with H&E stain. (a). Control rats: show the normal appearance of dermis and subdermis without any significantly lesion. (b).
Hemorrhage with moderately extravascular red blood cell and large amount of inflammatory leukocyte mainly neutrophils infiltration
in the subdermis interstitial tissue of mice following the subcutaneous injection of Carr only. Moreover, detail of the subdermis layer
show enlargement of the interstitial space caused by edema with exudates fluid. (c). Indomethacin (Indo) significantly reduced the level
of hemorrhage, edema and inflammatory cell infiltration compared to subcutaneous injection of Carr only. (d). Hispolon significantly show
morphological alterations compared to subcutaneous injection of Carr only. (100x) (e). The numbers of neutrophils were counted in each
scope (400x) and thereafter obtain their average count from five scopes of every tissue slice.∗∗P< .01, compared with Carr group.
superoxide and hydroxyl radicals, as well as the release of
other neutrophils-derived mediators . Some researches
demonstrate that inflammatory effect induced by Carr is
associated with free radical. Free radical, prostaglandin and
NO will be released when administrating with Carr for 1–
6h . The edema effect was raised to the maximum
at the third hour . Janero demonstrated that MDA
[37, 38]. Thus, inflammatory effect would result in the
accumulation of MDA. Glutathione is a known oxyradical
scavenger. Enhances the level of Glutathione conducive
toward favor reduces MDA the production. Cuzzocrea 
Evidence-Based Complementary and Alternative Medicine7
suggested that endogenous glutathione plays an important
role against Carr-induced local inflammation. In this study,
significantly increase in SOD, GRx and GPx activities with
hispolon treatment was found (Table 1). Furthermore, there
were significantly decreases in MDA level with hispolon
treatment (Figure 5). We assume that the suppression of
MDA production is probably due to the increase of SOD,
GRx and GPx activities.
In conclusion, these results suggested that hispolon
possessed analgesic and anti-inflammatory effects. The anti-
inflammatory mechanism of hispolon may be related to
iNOS and it is associated with the increase in the activities
of antioxidant enzymes (SOD, GPx and GRx). Hispolon may
be used as a pharmacological agent in the prevention or
treatment of disease in which free radical formation is a
China Medical University (CMU96-113 and CMU97-232);
National Science Council (NSC 97-2313-B-039 -001 -MY3).
H. Y. Chang and M. J. Sheu are contributed equally to this
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