1 Running title: Antioxidant, analgesic, and anti-inflammatory activities of the ethanolic
extracts of Taxillus liquidambaricola
Antioxidant, Analgesic, and Anti-inflammatory activities of the
ethanolic extracts of Taxillus liquidambaricola
Jeng-Shyan Denga, Chuan-Sung Chib,c, Shyh-Shyun Huangb, Pei-Hsin Shieb, Tsung-Hui Lind, Guan-Jhong Huangb,*
a
Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan
b
School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan.
c
Nursing Department, Hsin Sheng College of Medical Care and Management, Taoyuan 325, Taiwan
d
Department of Leisure, Recreation & Holistic Wellness, MingDao University, ChangHua 523, Taiwan
* Corresponding author:
Guan-Jhong Huang
School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan.
Tel: +886- 4- 2205-3366. Ext: 5508. Fax: +886- 4-2208-3362, E-mail address: [email protected]
2 1. Introduction
Inflammation is recognized as a biological process in response to tissue injury.
At the injury site, an increase in blood vessel wall permeability followed by migration
of immune cells can lead edema formation during inflammation. However, excessive
inflammation contributes to many acute and chronic human diseases (Rao et al., 2007).
Inflammatory response is characterized by the abundant productions of nitric oxide
(NO) and prostaglandin E2 (PGE2), and of cytokines, such as tumor necrosis factor
(TNF-), and thus, these pro-inflammatory mediators are important anti-inflammatory targets (Sheeba, and Asha 2009). Lipopolysaccharide (LPS) is an
endotoxin and a constituent of the outer membrane of gram-negative bacteria. LPS
stimulates innate immunity, by regulating the productions of inflammatory mediators,
like, NO, TNF-α, Interleukin-6, prostanoids, and leukotrienes (Liu, et al., 2007). And
in the animal the inflammation model of a carrageenan (Carr) induced edema is
usually used to assess the contributionof natural products in resisting the biochemical
changes associated with acute inflammation. Carr can induce acute inflammation
beginning with infiltration of phagocytes, the production of free radicals as wellas the
release of inflammatory mediators (Salvemini et al., 1996).
Intracellular antioxidant mechanisms against these inflammatory stresses involve
3 glutathione peroxidase (GPx) in tissues. Recently, it has been shown that faulty
cellular antioxidant systems cause organisms to develop a series of inflammatory and
cancer diseases (Valko et al., 2006). However, it appears that the various roles of
enzymatic antioxidants help to protect organisms from excessive generation of
oxidative stress in the inflammatory process, which has triggered studies focusing on
the role of natural products in suppressing the production of oxidation by increasing
enzymatic antioxidants in tissues (Huang et al., 2011).
Taxillus liquidambaricola (Hayata) Hosok, a parasitic plant that attacks the plant, which is called “Sang Ji Sheng” in Taiwan, and the whole plant (stems and leaves) has been traditionally used for the treatment of rheumatoid arthralgia, threatened
abortion and hypertension and also been applied as an anti-obesity herbal medicine
(Wang et al., 2008). Although Taxillus liquidambaricola has showed some
physiological effects, there are no studies focusing on its inhibitory effects on the
antioxidant, analgesic activities, and the mechanism of anti-inflammatory activities of
the ethanolic extracts of Taxillus liquidambaricola (ETL) in cell and animal models.
Consequently, the objective of the present study is to determine the therapeutical
4 2. Materials and methods
2.1. Materials
Lipopolysaccharide (LPS, Escherichia coli O127:B8), 1,1-Diphenyl-2-picrylhydrazyl
(DPPH), N-(1-naphthyl) ethylenediamine dihydrochloride, sulfanilamide, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), thiobarbituric acid (TBA), 3-[4,5-dimethyl-thiazol- 2-yl]-2,5-diphenyl tetrazolium bromide (MTT),
-Carrageenan (Carr), indomethacin (Indo), quercetin and other chemical reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA). Plant materials were
collected from Taichung country in Taiwan. They were identified and authenticated by
Dr. Yuan-Shiun Chang, Professor, School of Chinese Pharmaceutical Sciences and
Chinese Medicine Resources, College of Pharmacy, China Medical University.
2.2. Preparation of the extracts of plant materials
Dried sample of ETL (100 g) was macerated with 1L ethanol for 24 h at room
temperature. Filtration and collection of the extract was done three times. The filtrates
were collected, concentrated with a vacuum evaporator until the volume was below
5 2.3. Fingerprint chromatogram of ETL extracts by HPLC
The chromatographic system consisted of a Qaternary Gradient Pump SFD 2100,
a SFD 5200 autosampler, a Merck LiChrospher 100 RP-18e column (5 m, 4.0
I.D.×250 mm) and a S-3210 photodiode-array detector (PDA) (Schambeck SFD
GmbH, Bad Honnef, Germany). Peak area was calculated by using a Schambeck
HPLC-GPC-Software in the computer integrator. The samples were analyzed by
HPLC on a Lichrospher 100 RP-18e column and they were detected at 360 nm with
methanol / 0.5% phosphate solution (50: 50, v/v) as the mobile phase at a flow rate of
1.0 mL/min.
To the first sample (unhydrolyzed), 0.1 g of ETL was dissolved in 1 mL of
LC-grade methanol and filtered through ultra membrane filter (pore size 0.45 μm;
Millipore) before HPLC analysis. The second sample (hydrolyzed), 0.1 g of ETL, was
hydrolysis for 60 min in the presence of 8 mL 2% H2SO4 at 100°C heated in water
bath, efficiently released quercetin from quercetin glycosides, and partitioned five
times with 20 mL ethyl acetate. The ethyl acetate portions were combined, filtered and
then concentrated by a rotary evaporator and the residues dissolved in 1 mL of
LC-grade methanol and filtered through ultra membrane filter before HPLC analysis.
6
quercetin and quercetin glycosides. The separation of quercetin was carried out by
solvent partition and high performance liquid chromatography (HPLC). For the
identification of quercetin, photodiode-array detection was used.
2.4. In vitro antioxidant activities of crude extracts
2.4.1. Determination of antioxidant activity by DPPH radical scavenging ability
The effects of crude extracts and positive controls (BHT) on DPPH radicals were estimated according to the method of Huang et al., (2006). Aliquot (20 μL) of crude extracts at various concentrations were each mixed with 100 mM Tris-HCl buffer (80 μL, pH 7.4) and then with 100 μL of DPPH in ethanol to a final concentration of 250 μM. The mixture was shaken vigorously and left to stand at room temperature for 20 min in the dark. The absorbance of the reaction solution was measured
spectrophotometrically at 517 nm. The percentages of DPPH decolorization of the
samples were calculated according to the equation: % decolorization = [1- (ABS sample
/ABS control)] ×100. EC50 value was the effective concentration at which DPPH
radicals were scavenged by 50% and was obtained by interpolation from linear
7 2.4.2. Determination of antioxidant activity by ABTS·+ scavenging ability
The ABTS·+ scavenging ability was determined according to the method of
Huang et al., (2006). Aqueous solution of ABTS (7 mM) was oxidized with potassium
peroxodisulfate (2.45 mM) for 16 hrs in the dark at room temperature. The ABTS·+
solution was diluted with 95% ethanol to an absorbance of 0.75 ± 0.05 at 734 nm
(Beckman UV-Vis spectrophotometer, Model DU640B). An aliquot (20 μL) of each
sample (125 μg/mL) was mixed with 180 μL ABTS·+ solution and the absorbance was
read at 734 nm after 1 min. Trolox was used as a reference standard.
2.5. Determination of total polyphenol content
The total polyphenol contents of crude extracts were determined according to the
method of Huang et al (2008). 20 μL of each extract (125 μg/mL) was added to 200 μL distilled water and 40 μL of Folin-Ciocalteu reagent. The mixture was allowed to stand at room temperature for 5 min and then 40 μL of 20 % sodium carbonate was added to the mixture. The resulting blue complex was then measured at 680 nm.
Catechin was used as a standard for the calibration curve. The polyphenol content was
calibrated using the linear equation based on the calibration curve. The total
8 2.3. Cell culture
A murine macrophage cell line RAW 264.7 (BCRC No. 60001) was purchased
from the Bioresources Collection and Research Center (BCRC) of the Food Industry
Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in plastic dishes containing Dulbecco‟s Modified Eagle Medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, USA) in a CO2
incubator (5% CO2 in air) at 37°C and subcultured every 3 days at a dilution of 1:5
using 0.05% trypsin–0.02% EDTA in Ca2+-, Mg2+- free phosphate-buffered saline
(DPBS).
2.3.1. Cell viability.
Cells (2 x 105) were cultured in 96-well plates containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. Then cells were cultured with samples in the presence of 100 ng/mL LPS for 24 hrs. After that, the cells were washed twice with DPBS and incubated with 100 L of 0.5 mg/mL MTT for 2 hrs at 37°C testing for cell viability. The medium was then discarded and 100 L dimethyl sulfoxide (DMSO) was added. After 30-min incubation, absorbance at 570 nm was read using a microplate reader.
2.3.2. Measurement of Nitric oxide/Nitrite
Nitrite levels in the cultured media and serum, which reflect intracellular NO synthase activity, were determined by Griess reaction (Huang et al., 2007). The cells
9 were incubated with samples in the presence of LPS (100 ng/mL) at 37°C for 24 hrs. Then, cells were dispensed into 96-well plates, and 100 L of each supernatant was mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 5% phosphoric acid) and incubated at room temperature for 10 min. Using sodium nitrite to generate a standard curve, the concentration of nitrite was measured form absorbance at 540 nm.
2.4. Animals
This study was conducted in conformity with the policies and procedure details in the “Guide for the Care and Use of Laboratory Animals” (NIH Publication No. 86–23 1985) and was approved by the ethics committee of the Institutional Animal
Care and Use Committee (IACUC) of China Medical University, Taichung, Taiwan.
ICR strain male mice (6−8 weeks old) were obtained from BioLASCO Taiwan Co.,
Ltd., Taipei, Taiwan. The animals were housed in an environmentally controlled room
(temperature 22 ± 1 °C; relative humidity 55 ± 5%; 12 h dark–light cycle). They were
given food and water ad libitum.
After a 2-week adaptation period, male ICR mice (18-25 g) were randomly
assigned to five groups (n=6) of the animals in acetic acid-induced writhing (1%, 0.l
10 These include a pathological model group (received acetic acid or formalin), a
positive control (acetic acid or formalin + Indo), and ETL administered groups (acetic
acid or formalin+ ETL: 0.25, 0.5, and 1.0 g/Kg). In the Carr-induced edema
experiment, there were randomly assigned to six groups (n=6) of the animals in the
study. The control group receives normal saline (i.p.). The other five groups include a
Carr-treated, a positive control (Carr + Indo) and ETL administered groups (Carr +
ETL: 0.25, 0.5, and 1.0 g/Kg).
2.4.1. Acetic acid-induced writhing response
After a 2-week adaptation period, male ICR mice (18 to 25 g) were randomly
assigned to six groups (n = 8) including a normal control, an Indo positive control and
four ETL-treated groups. Control group received 1% acetic acid (10 mL/Kg body
weight) and the positive control group received Indo (10 mg/Kg, i.p.) 25 min before
intraperitoneal injection of 1% acetic acid (10 mL/Kg body weight). ETL-treated
groups received ETL (0.25, 0.5, and 1.0 g/Kg, p.o.) 55 min before intraperitoneal
injection of 1% acetic acid (10 mL/Kg body weight). Five minutes after the i.p.
injection of acetic acid, the number of writhing during the following 10 minutes was
11 2.4.2. Formalin test
The antinociceptive activity of the drugs was determined using the formalin test
(Dubuisson and Dennis, 1977). Control group received 5% formalin. Twenty
micro-liter of 5% formalin was injected into the dorsal surface of the right hind-paw
60 min after administration of ETL (0.25, 0.5, and 1.0 g/Kg, p.o.) and 30 min after
administration of Indo (10 mg/Kg, i.p.). The mice were observed for 30 min after the
injection of formalin, and the amount of time spent licking the injected hind paw was
recorded. The first 5 min post formalin injection is referred to as the early phase and
the period between 15 min and 40 min as the late phase. The total time took licking or
biting the injured paw (pain behavior) was measured with a stop watch. The activity
was recorded in 5 min intervals.
2.4.3. Determination of carrageenan (Carr) induced edema
Carr-induced hind paw edema model was used for determination of
anti-inflammatory activity (Winter et al., 1962). After a 2-week adaptation period,
male ICR mice (18 to 25 g) were randomly assigned to five groups (n = 6) including
Carr, positive Indo control and three ETL-treated groups. Carr group received 1%
Carr (50 μL). ETL at doses of 0.25, 0.5, and 1.0 g/Kg were orally administered 2 hrs before the injection with 1% Carr (50 μL) in the plantar side of right hind paws of the
12 mice. And Indo (10 mg/Kg) was intraperitoneally administered 90 min before the
injection with 1% Carr (50 μL) in the plantar side of right hind paws of the mice. Paw
volume was measured immediately after Carr injection at 1, 2, 3, 4, and 5 h intervals
using a plethysmometer (model 7159, Ugo Basile, Varese, Italy). The degree of
swelling induced was evaluated by a minus b, where a was the volume of the right
hind paw after Carr treatment and b was the volume of the right hind paw before Carr
treatment. Indo was used as a positive control.
In the later experiment, the right hind paw tissue was taken at the 5th h. The right
hind paw tissue was rinsed in ice-cold normal saline, and immediately placed in cold
normal saline four times their volume and homogenized at 4 ºC. Then the homogenate
was centrifuged at 12,000×g for 5 min. The supernatant was obtained and stored at −20 ºC refrigerator for MDA and the antioxidant enzymes (CAT, SOD, and GPx) activities assays.
2.4.4. Determination of tissue lipid peroxidation
MDA was evaluated by the thiobarbituric acid reacting substances (TRARS)
method (Ohishi et al., 1985). Briefly, MDA reacted with thiobarbituric acid in the
acidic high temperature and formed a red-complex TBARS. The absorbance of
13 2.4.6. Measurement of tumor necrosis factor (TNF-α) in serum
Serum levels of TNF-α were determined using a commercially available ELISA
kit (Biosource International, Inc., Camarillo, CA) according to the instructions of the
manufacturer. TNF-α was determined from a standard curve.
2.4.7. Determination of antioxidant enzyme activity in paw tissue
The following biochemical parameters were analyzed to check the protective
activity of ETL by the methods given below. Total SOD activity was determined by
the inhibition of cytochromec reduction (Flohe and Otting 1984). The reductionof
cytochrome c was mediated by superoxide anions generatedby the xanthine/xanthine
oxidase system and monitored at 550 nm.One unit of SOD was defined as the amount
of enzyme requiredto inhibit the rate of cytochrome c reduction by 50%. Total CAT
activity estimation was based on the previously reported (Armstrong & Browne,
1994). In brief, the reduction of 10 mM H2O2 in 20 mM of phosphate buffer (pH 7)
was monitored by measuring the absorbance at 240 nm. The activity was calculated
by using a molar absorption coefficient, and the enzyme activity was defined as
14 GPx activity in cytosol was determined as previously reported (Flohe & Gunzler,
1984). The enzyme solution was added to a mixture containing hydrogen peroxide
and glutathione in 0.1 mM Tris buffer (pH 7.2) and the absorbance at 340 nm was
measured. Activity was evaluated from a calibration curve, and the enzyme activity
was defined as nanomoles of NADPH oxidized per milligram protein per minute. The
protein concentration of the tissue was determined by the Bradford dye-binding assay
(Bio-Rad, Hercules, CA).
2.4.8. Histological examination
For histological examination, biopsies of paws were taken 5th hrs following the
interplanetary 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 5 μm) were
deparaffinized with xylene and stained with H & E stain. All samples were observed
and photographed with BH2 Olympus microscopy. Every 3~5 tissue slices were
randomly chosen from Carr, Indo and ETL treated (1.0 g/Kg) groups. The numbers of
neutrophils were counted in each scope (400 x) and thereafter obtain their average
15 2.4.9. Protein Lysate Preparation and Western blot Analysis of iNOS and COX-2
Total protein was extracted with a RIPA solution (radioimmuno-precipitation assay
buffer) at -20°C overnight. We used BSA (bovine serum albumin) as a protein
standard to calculate equal total cellular protein amounts. Protein samples (30g)
were resolved by denaturing sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) using standard methods, and then were transferred to
PVDF membranes by electroblotting and blocking with 1% BSA. The membranes
were probed with the primary antibodies (iNOS, COX-2, and -actin) at 4°C
overnight, washed three times with PBST, and incubated for 1 h at 37 °C with
horseradish peroxidase conjugated secondary antibodies. The membranes were
washed three times and the immunoreactive proteins were detected by enhanced
chemiluminescence (ECL) using hyperfilm and ECL reagent (Amersham
International plc., Buckinghamshire, U.K.). The results of Western blot analysis were
quantified by measuring the relative intensity compared to the control using Kodak
Molecular Imaging Software and represented in the relative intensities.
2.5. Statistical analysis
Data are expressed as mean ± S.E. Statistical evaluation was carried out by
16 Statistical significance is expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.
17
3. Results
3.1 Fingerprint Analysis by HPLC
To establish the fingerprint chromatogram for the quality control of ETL,
quercetin was used as markers. Quercetin rarely occurs in the free state but usually
present as O-glycosides and are linked to sugars like glucose, galactose or rhamnose.
Determination of quercetin presented as glycosides and aglycone forms in ETL for
quality assessment. To accurately determinate the quercetin and quercetin glycosides,
the glycosyl groups on the quercetin glycosides should be removed by acid hydrolysis
and converted to quercetin (aglycone) before HPLC. An optimized HPLC-DAD
technique was employed. According to the plot of peak-area ratio (y) vs.
concentration (x, μg/mL), the regression equations and correlation coefficient (r) was
y = 0.094x + 0.033 (r2=0.9992). Fig. 1 shows HPLC fingerprint chromatograms of
quercetin (Fig. 1A), ETL (Fig. 1B), and ETL after acid hydrolysis (Fig. 1C).
Quercetin component has been identified as quercetin by the retention time (29.8 min)
and UV absorbance of purified standard. The extract without hydrolysis contained
only traces of free quercetin (10.5 μg/g dry weight). The content of quercetin after
hydrolysis in ETL was consistently high (126.6 μg/g dry weight) and comparable with
the content before hydrolysis. The aglycone form of quercetin accounts for only about
8% of the total quercetin content. A significant amount of quercetin was found in
18
samples, suggesting that quercetin exists in combined forms.
3.1. The contents of phytochemicals extracted and the antioxidant activities of ETL.
Plants containing polyphenols have been reported to possess strong antioxidant
activities (Hung et al., 2006). The results showed that ETL had the highest phenolic
contents of 352.31 ± 2.68 g CE/mg, respectively (Table 1). Total flavonoid content
was expressed as mg of rutin equivalent per gram of dry weight. As shown in Table 1,
the total flavonoid content of ETL was 38.48± 1.38 g RE/mg.
Table 1 also shows ABTS and DPPH scavenging activities of ETL. TEAC value
of ETL was 1063.53 ± 6.34 g/mg. And ETL exhibited the strongest antioxidant
activities in scavenging DPPH radicals, with EC50 values of 88.72 ± 3.57 g/mL,
respectively. We also evaluated the reference compound of quercetin exhibited the
strongest antioxidant activities in ABTS and DPPH scavenging radicals in the ETL.
3.2. Effect of the ETL on LPS-induced NO Production in Macrophages
In a cellular model of inflammation, the NO inhibitory activity of ETL was
determined by using the LPS activated macrophages to produce NO radicals that were
19 ETL reduced the NO production of activated macrophages with an IC50 value of
386.38 ± 2.54 g/mL, respectively. This suggests ETL could be a potential inhibitor of NO related inflammation pathway. In addition, no cell toxicity was observed with
ETL (0, 250, 500, and 1000 g/mL), as measured by the MTT cell viability test. And
the reference compounds of quercetin (0, 5, 10, and 20 ) in the ETL also showed
the NO inhibitory activity induced by LPS in RAW264.7 macrophages with an IC50
value of 16.42 ± 0.21g/mL, respectively (Fig. 2 and Table 1).
3.3. Inhibition of LPS-induced iNOS and COX-2 Protein by ETL and Quercetin.
The results showed that incubation with ETL and quercetin in the presence of LPS
for 24 hrs inhibited iNOS and COX-2 protein expression in mouse macrophage
RAW264.7 cells in a dose-dependent manner (Fig. 3A). The intensity of protein bands
were analyzed and showed an average of 73.8% and 76.2% down-regulation of iNOS
and COX-2 proteins, respectively, after treatment with ETL at 1000 g/mL compared
with the LPS-alone (Fig. 3B). And the protein expression showed an average of
78.8%, and 19.2% down-regulation of iNOS and COX-2 protein after treatment with
quercetin at 20 (Fig. 3A). The down-regulation of iNOS and COX-2 activity of
ETL (1000g/mL) was better than quercetin (20 ).
20 The cumulative amount of abdominal stretching correlated with the level of
acetic acid-induced pain (Fig. 4A). ETL treatment (0.25, 0.5, and 1.0 g/Kg)
significantly inhibited the number of writhing in comparison with the pathological
model group. The inhibition rates of the number of writhing compared with the
pathological model group are 22.84%, 40.08%, and 57.78% respectively. The
inhibiting effect of acetic acid-induced writhing by ETL (1.0 g/kg) was similar to
that produced by a positive control Indo (10 mg/kg) (P < 0.001).
3.2.2. Formalin test
ETL significantly inhibited formalin-induced pain in the late phase; however,
there was no inhibition in the early phase (Fig. 1B). ETL treatment (0.25, 0.5, and 1.0
g/Kg) significantly inhibited the formalin-induced pain (late phase) in comparison
with the pathological model group. The inhibition rates of formalin-induced licking
compared with the pathological model group are 30.53%, 43.78%, and 52.55%,
respectively. This inhibiting effect of formalin-induced licking time by ETL (1.0 g/kg;
P < 0.001) was better than a positive control Indo (10 mg/kg) (P < 0.001).
3.2.3. -Carrageenan (Carr)-induced edema
21 anti-inflammatory drug used to reduce acute inflammatory response such as swelling.
According to Fig. 5A, Indo (10 mg/Kg) reduced the edema volumes about 53.3% in
comparison to the Carr group during the 5th h of Carr treatment. Further, in the range
of 0.25–1.0 g/Kg, ETL showed a concentration dependent inhibition of edema
development. For ETL at the concentration of 1.0 g/Kg, the levels of edema volume
were decreased to 49.5% of that observed in the Carr group after 5th h treatment. These
data imply that ETL can exhibit an inhibitor of edema in acute inflammatory
processes.
3.2.4. Effects of ETL on MDA, NO, and TNF- levels
Lipid oxidation serves as a marker of cellular damage and has been recognized as
a marker of inflammatory damage. As shown in Fig. 5B, Carr increased the level of
lipid oxidation by 3.57 folds in comparison with the control group. Meanwhile, Indo
decreased the level of lipid oxidation to 51.2% of that observed in the Carr group. In
fact, in the range of 0.25-1.0 g/Kg, ETL inhibited the level of lipid oxidation down to
0.27-54.4% of that observed in the Carr group. These data imply that ETL can protect
against tissue lipid oxidation in Carr induced inflammatory processes.
Many studies demonstrated that Carr-induced inflammatory processes increased
22 regular index for intracellular NO and iNOS production in vivo. As shown in Fig. 5C,
Carr increased the level of nitrite by 9.2 folds in comparison to the control group in
serum. Meanwhile, Indo decreased the level of serum nitrite to 56.7% of that observed
in the Carr group. In fact, in the range of 0.25-1 g/Kg, ETL reduced the level of nitrite
to 29.5-61.5% of that observed in the Carr group. And Carr increased the level of
TNF- in the serum by 5.6 folds in comparison to the control group (Fig. 5D). Indo
decreased the level of serum TNF- to 58.5% of that observed in the Carr group.
ETL also inhibited the production of TNF- to 15.3-61.9% of that observed in the
Carr group. These data imply that ETL acts as an inhibitor of Carr induced tissue
inflammation by decreasing NO and TNF- production in vivo.
3.2.5. Effects of ETL on the activities of antioxidant enzymes in Carr-induced paw edema
Under healthy conditions, free radicals are prevented by enzymes directly
interacting with ROS. Table 2 shows the activities of CAT, SOD, and GPx in
Carr-induced paw edema of treated mice. Carr decreased the activities of CAT, SOD,
and GPx in Carr-induced paw edema by 41.1%, 56.4%, and 50.3% respectively, in
comparison to the control group (p<0.001). In the range of 0.25-1.0 g/Kg, ETL
23 to 133.5%-187.4% respectively, compared to that observed in the Carr group. Indo
also exhibited increase effects in the activities of CAT (147.8%), SOD (194.1%), and
GPx (174.8%) in comparison to the Carr group. These data imply that the
anti-inflammatory effects of ETL in vivo might be attributed to its elevation in the
antioxidant enzymes activities of Carr-induced mice.
3.2.6. Effects of ETL on Carr-induced iNOS and COX-2 protein expressions in Mice
Paw Edema
The results showed that administered of ETL (1.0 g/Kg) on Carr-induced for 5th h
inhibited iNOS and COX-2 proteins expression in mouse paw edema (Fig. 6A). The
intensity of protein bands was analyzed and showed an average of 57.6% and 72.4%
down-regulation of iNOS and COX-2 protein (p < 0.001) respectively, after treatment
with ETL at 1.0 g/Kg compared with the Carr-induced alone (Fig. 6B). In addition,
the protein expression showed an average of 47.4% and 49.1% down-regulation of
iNOS and COX-2 protein after treatment with Indo at 10 mg/Kg compared with the
Carr-induced alone.
3.2.7. Histological examination.
24 inflammatory response Carr-induced. Actually inflammatory cells were reduced in
number and were confined to near the vascular 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 (Fig. 7A). Neutrophils were notified increased with Carr treatment (p <
0.001). Indo and ETL (1.0 g/Kg) could significantly decrease the neutrophils numbers
25
4. Discussion
Free radicals could play an important role in the degenerative or pathological
processes of various serious diseases, such as aging, cancer, coronary heart disease, Alzheimer‟s disease, neurodegenerative disorders, atherosclerosis, cataracts, and inflammation (Hung et al., 2006). The use of traditional medicine is widespread and
plants still present a large source of natural antioxidants that might serve as leads for
the development of novel drugs. The higher radical scavenging activity of ETL seems
to be closely correlated with its polyphenolic constituents though active components
could play important roles in its antioxidative effect. Consequently, it is possible that
the total phenolic constituents may contribute to anti-inflammatory activity of ETL. In
this paper, we demonstrated that ETL inhibited radical scavenging and NO production.
And the reference compound of quercetin in the ETL also with the antioxidant and
anti-inflammatory activities (Table 1).
Triterpenoid, flavonoids, and phenolic acids possessed analgesic and
anti-inflammatory effects on animal models and the pharmacological effects (Arslan
et al., 2010). Studies have also demonstrated that flavonoids such as rutin, quercetin,
luteolin produced significant antinociceptive and anti-inflammatory activities
(Deliorman et al., 2007). Hence, it was suggested that the antioxidant, analgesic, and
26
analgesic testing methods were employed with the objective of identifying possible peripheral and central effects of the test substances. The acetic writhing test
is used to study the peripheral analgesic effects of drugs (Koster et al., 1959). Related
studies have demonstrated that acetic acid indirectly induces the release of
endogenous mediators of pain that stimulate the nociceptive neurons, which are
sensitive to nonsteroidal anti-inflammatory drugs (Arslan et al., 2010). When
compared antinociceptive activities, ETL was relatively potent in acetic acid writhing
test indicating peripheral antinociception. In contrast, ETL (1.0 g/Kg) exhibited an
action in similar magnitude with Indo, a reference drug for peripheral antinociception
(Fig. 4A). Formalin-induced paw pain produced a distinct biphasic nociception, a first
phase (lasting the first 5 min) corresponding to acute neurogenic pain, and a second
phase (lasting from 15 to 30 min) corresponding to inflammatory pain responses
(Huang et al., 2011). Therefore, the test can be used to clarify the possible mechanism
of an antinociceptive effect of a proposed analgesic. The inhibitory effect of ETL on
the nociceptive response in the late phase of the formalin test suggested that the
anti-nociceptive effect of ETL could be due to its peripheral action (Fig. 4B).
Carr-induced inflammation has been well established as a valid model to study
free radical generation in paw tissue after inflammatory states. The cellular and
27 these models of inflammation are standard models of screening for anti-inflammatory
activity of various experimental compounds (Kumar and Kuttan, 2009). It appears that
the early phase of the Carr edema is related to the production of histamine,
leukotrienes and possibly cyclooxygenase products, while the delayed phase of the
Carr-induced inflammatory response has been linked to neutrophil infiltration and the
production of neutrophil-derived free radicals, such as hydrogen peroxide, superoxide
and OH radicals, as well as to the release of other neutrophil-derived mediators. The
degree of paws swelling was maximal at 3th hrs after injection of Carr. However, a
reduction in paw swelling size is a good index in determining the protective action of
anti-inflammatory agents. According to Fig. 5A, ETL (1.0 g/Kg) inhibited the
development of edema at 5th hrs after treatment. And quercetin also administered
before Carr clearly blocked Carr-induced inflammation in the rats (Morikawa et al.,
2003).
In the process of inflammation, a burst of NO is synthesized from L-arginine by
iNOS in activated macrophages. In fact, the overproduction of NO could induce cell
damage as well as inflammation. Our data imply that the inhibitory effects of ETL on
NO production could contribute to the decrease of oxidative stress and inflammation
development in tissues. It has been proposed that free radicals play an important role
28 produced by activated macrophages also plays an important mediator in the
cytotoxic/cytostatic mechanism of non-specific immunity. Therefore, ETL decreased
NO production in vitro (Table 1) and in vivo (Fig. 2B), which could further lead to
reduce the edema response in inflammation.
During inflammatory processes, large amounts of the proinflammatory mediators,
NO and PGE2, are generated by inducible iNOS and COX-2, respectively. INOS, is
generally not present in resting cells, but is induced by various stimuli, which include
bacterial LPS, TNF-α, IL-1β and interferon-γ (Salvemini et al., 2003). However,
COX-2 is induced by pro-inflammatory stimuli, including LPS and cytokines in cells
in vitro and in inflamed sites in vivo. In this study, there is a significant decrease in
iNOS and COX-2 activities with ETL treatment (Fig. 3A and 6A). We assume the
suppression of NO production is probably due to the decreases of iNOS and COX-2
activities. Moreover, ETL act as herbal antioxidants and its antioxidative action may
partly be responsible for the inhibition of NO production. Therefore, the inhibitory
effect of ETL on NO production could be contributed to its total polyphenols
inhibition to iNOS protein expression. Quercetin also exerts its anti-inflammatory
property by suppressing NO production and iNOS through the inhibition of
extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein
29 pathways (Cho, et al 2003).
Lipid oxidation not only serves as a marker of cellular damage in vivo but also
has been recognized to be the inducer of inflammatory processes. Some researches
demonstrate that inflammatory effect induced by Carr is associated with free radicals.
Free radicals, prostaglandin and NO will be released when administrating with Carr
for 1-6 hrs (Huang et al., 2011). MDA production is due to free radical attack plasma
membrane. Thus, inflammatory effect would result in the accumulation of MDA. In
this study, ETL not only exhibited radicals scavenging capacity and could decrease
Carr induced lipid damage in vivo (Fig. 5B).
In a number of pathophysiological conditions associated with inflammation or
oxidant stress, these ROS have been proposed to mediate cell damage via a number of
independent mechanisms including the inactivation of a variety of antioxidant
enzymes. Giving the importance of the oxidative status in the formation of edema, the
anti-inflammatory effect exhibited by drug in this model might be related to its
antioxidant properties (Bignotto et al., 2009). The role of CAT is to decompose H2O2.
Increased SOD activity can protect cells against threat of reactive free radicals. GPx is
regarded as a crucial enzyme which catalyses the reduction of hydroperoxide. As
shown in Table 2, there was a significant increase in CAT, SOD, and GPx activities
30 due at least in part to elevate intracellular antioxidant enzyme activities and decrease
inflammatory stress in tissue. However, we found that ETL decreased radical
production and lipid oxidation in vitro as well as in vivo. These data suggest that ETL
could serve as a natural antioxidant to protect cells against inflammatory damage.
In conclusion, our data suggest that ETL shows anti-inflammatory effects in vitro
and in vivo. The anti-inflammatory effects of ETL may be related to iNOS and COX-2
reduction and reduce excess TNF- generation in physiological systems. The
antioxidant effects of ETL can be due to increase in the activities of antioxidant
enzymes and its effects on radicals scavenging. Therefore, we suggest that ETL
contain herbal antioxidants and exhibit analgesic, anti-inflammatory activity in vivo.
Acknowledgement
The authors want to thank the financial supports from the National Science Council
(NSC 97-2313-B-039 -001 -MY3), China Medical University (CMU) (CMU99-S-29,
CCM-P99-RD-042, and CMU99-COL-10) and Taiwan Department of Heath Clinical
31
References
Armstrong, D., Browne, R., 1994. The analysis of free radicals, lipid peroxides,
antioxidant enzymes and compounds related to oxidative stress as applied to the
clinical chemistry laboratory. Advances in Experimental Medicine and Biology 366, 43-58.
Arslan, R., Bektas, N., Ozturk, Y., 2010. Antinociceptive activity of methanol extract
of fruits of Capparis ovata in mice. Journal of Ethnopharmacology,131, 28-32.
Bignotto, L., Rocha, J., Sepodes, B., Eduardo-Figueira, M., Pinto, R., Chaud, M., de
Carvalho, J., Moreno, H., Mota-Filipe, H. Anti-inflammatory effect of lycopene
on carrageenan-induced paw oedema and hepatic ischaemia-reperfusion in the
rat. British Journal of Nutrition 102, 126-33.
Cho, S.Y., Park, S.J., Kwon, M. J., Jeong, T.S., Bok, S.H., Choi, W.Y., Jeong, W.I.,
Ryu, S.Y., Do, S.H. Lee, C.S., Song, J. C., Jeong, K.S., 2003. Quercetin
suppresses proinflammatory cytokines production through MAP kinases and
NF-κB pathway in lipopolysaccharide-stimulated macrophage. Molecular and
Cellular Biochemistry 243, 153–160.
Deliorman, O.D., Hartevioğlu, A., Küpeli, E., Yesilada, E., 2007. In vivo
anti-inflammatory and antinociceptive activity of the crude extract and fractions
32 Dubuisson, D., Dennis, S.G., 1977. The formalin test: A quantitative study of the
analgesic effects of morphine, meperidine, and brain stem stimulation in rats
and cats. Pain 4, 161-174.
Flohe, L., Gunzler, W.A., 1984. Assays of glutathione peroxidase. Methods in
Enzymology 105, 114-121.
Flohe, L., Otting, F., 1984. Superoxide dismutase assays. Methods in Enzymology
105, 93-104.
Huang, M.H., Huang, S.S., Wang, B.S., Wu, C.H., Sheu, M.J., Hou, W.C., Lin, S.S.,
Huang, G.J., 2011. Antioxidant and anti-inflammatory properties of
Cardiospermum halicacabum and its reference compounds ex vivo and in vivo.
Journal of Ethnopharmacology 133, 743-750.
Huang, D.J., Chen, H.J., Hou, W.C., Lin, Y.H., 2006. Sweet potato (Ipomoea batatas [L.] Lam „Tainong 57‟) storage roots mucilage with antioxidant activities in vitro. Food Chemistry 98, 774-781.
Huang, S.S., Huang, G.J., Ho, Y.L., Lin, Y.H., Hung, H.J. Chang, T.N., Chan, M.J.,
Chen, J.J., Chang, Y.S., 2008.Antioxidant and antiproliferative activities of the
33 Huang, G.J., Sheu, M.J., Chen, H.J., Chang, Y.S., Lin, Y.H., 2007. Inhibition of
Reactive Nitrogen Species in Vitro and ex Vivo by Trypsin Inhibitor from sweet
potato 'Tainong 57' storage roots. Journal of Agricultural and Food Chemistry
55, 6000-6006.
Huang, G.J., Huang, S.S., Lin, S.S., Shao, Y.Y., Chen, C.C., Hou, W.C. Kuo, Y.H.,
2010. Analgesic Effects and the Mechanisms of Anti-inflammation of
Ergostatrien-3 beta-ol from Antrodia camphorata Submerged Whole Broth in
Mice. Journal of Agricultural and Food Chemistry 58, 7445-7452.
Hung, T.M., Na, M.K., Thuong, P.T., Su, N.D., Sok, D.E., Song, K.S., Seong, Y.H.,
Bae, K.H., 2006. Antioxidant activity of caffeoyl quinic acid derivatives from
the roots of Dipsacus asper Wall. Journal of Ethnopharmacology 108, 188-192.
Huang, S.S., Chiu, C. S., Chen, H. J., Lin, S. S., Hsieh, I.C., Hou, W.C., Huang, G.J.,
2011. Antinociceptive activities and the mechanisms of anti-inflammation of
asiatic acid in mice. Evidence Based Complementary and Alternative Medicine
doi:10.1155/2011/895857.
Koster R, Anderson M, De BEJ. 1959. Acetic acid for analgesic screening. Federation
Proceeding 18, 412-416.
34 regulates nitric oxide and proinflammatory cytokines profile in carrageenan
induced paw edema model. Immunopharmacology and Immunotoxicology 31,
94-102.
Liu, D.Z., Liang, H.J., Chen, C.H., Su, C.H., Lee, T.H., Huang, C.T., Hou, W.C., Lin,
S.Y., Zhong, W.B., Lin, P.J., Hung, L.F., Liang, Y.C., 2007. Comparative
anti-inflammatory characterization of wild fruiting body, liquid-state
fermentation, and solid-state culture of Taiwanofungus camphoratus in
microglia and the mechanism of its action. Journal of Ethnopharmacology 113,
45–53.
Morikawa, K., Nonaka, M., Narahara, M., Torii, I., Kawaguchi, K., Yoshikawa, T.,
Kumazawa, Y., Morikawa, S., 2003. Inhibitory effect of quercetin on
carrageenan-induced inflammation in rats. Life Sciences 74, 709–721.
Ohishi, N., Ohkawa, H., Miike, A., Tatano, T., Yagi, K., 1985. A new assay method
for lipid peroxides using a methylene blue derivative. Biochemistry
International 10, 205-211.
Rao, Y.K., Fang, S.H., Tzeng, Y.M., 2007. Evaluation of the anti-inflammatory and
anti-proliferation tumoral cells activities of Antrodia camphorata, Cordyceps
sinensis, and Cinnamomum osmophloeum bark extracts. Journal of
35 Salvemini, D., Wang, Z.Q., Bourdon, D.M., Stern, M.K., Currie, M.G. and Manning,
P.T., 1996. Evidence of peroxynitrite involvement in the carrageenan-induced
rat paw edema. European Journal of Pharmacology 303, 217-220.
Salvemini, D., Ischiropoulos, H., Cuzzocrea, S., 2003. Roles of nitric oxide and
superoxide in inflammation. Methods in Molecular Biology 225, 291–303.
Sheeba, M.S., Asha, V.V., 2009. Cardiospermum halicacabum ethanol extract
inhibits LPS induced COX-2, TNF-alpha and iNOS expression, which is
mediated by NF-kappa B regulation, in RAW264.7 cells. Journal of
Ethnopharmacology 124, 39-44.
Valko, M., Rhodes, C.J., Moncol, J., Izakovic, M. and Mazur, M., 2006. Free radicals,
metals and antioxidants in oxidative stress-induced cancer. Chemico-Biological
Interactions 160, 1-40.
Wang, Y., Deng, M., Zhang, S.Y., Zhou, Z.K., Tian, W.X., 2008. Parasitic loranthus
from Loranthaceae rather than Viscaceae potently inhibits fatty acid synthase
and reduces body weight in mice. Journal of Ethnopharmacology 118, 473–478.
Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenin-induced edema in hind
paw of the rat as an assay for antiiflammatory drugs. Proceedings of the Society
36 Medicine (New York, N.Y.) 111, 544-547.
