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Running Title: Chemical characterization and anti-inflammatory activities of Actinidia callosa var. ephippioides in vivo

Chemical characterization and in vivo anti-inflammatory activities of Actinidia callosa var. ephippioides via suppression of proinflammatory cytokines

Jung-Chun Liaoa,#, Shyh-Shyun Huanga,#, Jeng-Shyan Dengb, Chao-Ying Leea, Ying-Chih Linc, Guan-Jhong Huangd, *
aSchool of Pharmacy, College of Pharmacy, China Medical University, Taichung 404, Taiwan

bDepartment of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan

cDepartment of Optometry, Jen-Teh Junior College of Medicine, Nursing and Management,Miaoli 356, Taiwan

dDepartment of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan
*Corresponding author

Guan-Jhong Huang

Department 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:
ΔJung-Chun Liao and Shyh-Shyun Huang contributed equally to this work.


Actinidia callosa var. ephippioides (ACE) has been widely used to treat anti-pyretic, antinociceptive, anti-inflammation, abdominal pain, and fever in Taiwan. This study aimed to determine the mechanism of anti-inflammatory activities of ethyl acetate fraction of ACE (EA-ACE) using model of λ-carrageenan (Carr)-induced paw edema in mouse model. In HPLC analysis, chemical characterization of EA-ACE was established. In order to investigate the anti-inflammatory mechanism of EA-ACE, we have detected the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) and the levels of malondialdehyde (MDA) in the paw edema. Serum NO, tumor necrosis factor (TNF-α), and interleukin-1 (IL-1βwere evaluated. Chemical characterization from HPLC indicated that EA-ACE contains betulinic acid, ursolic acid, and oleanolic acid. In the anti-inflammatory test, EA-ACE decreased the paw edema after Carr administration, increased the activities of CAT, SOD, and GPx and decreased the MDA level in the edema paw at the 5th hr after Carr injection. EA-ACE affects the serum NO, TNF-α, and IL-1β levels at the 5th hr after Carr injection. EA-ACE decreased Carr-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions by Western blotting. Actinidia callosa var. ephippioides have the potential to provide a therapeutic approach to inflammation-associated disorders.

Key words: Actinidia callosa var. ephippioides; Anti-inflammation; MDA; NO; TNF; IL-1β


Actinidia callosa var. ephippioides (Actinidiaceae; ACE) is used as a folk medicine in Taiwan. The stems of the plant have been extensively employed to treat various ailments like leprosy, abscess, rheumatism, arthritis inflammation, jaundice, and abnormal leucorrhea and were also useful for the treatment of cancers, especially those of lung, liver, and digestive system (Gan, 1993). Actinidia species also had showed some pharmacological effects such as anti-inflammatory activity from the fruit of Actinidia polygama (Kim et al., 2003), hypolipidemic effects from the leaves of Actinidia kolomikta (Zhao et al., 2012), antitumor, and immunomodulatory activity from the roots of Actinidia eriantha (Xu et al., 2009). However, no studies have been conducted to investigate the antinociceptive and anti-inflammatory activities of ACE in mice.

Pain is a common situation and is one of the most frequently observed symptoms of different pathologies. The principal targets of effective pain control are to ameliorate nociception, to reduce threshold of pain sensation, and to improve quality of life.  Although there are a great number of analgesic opioid and nonopioid drugs, there is some concern regarding the safety and side effects of these drugs, such as ulcers, bleeding, and dependence, which limit their clinical use (Jage, 2005).

Inflammation is recognized as a biological process in response to tissue injury. At the tissue injury site, an increase in blood vessel wall permeability followed by migration of immune cells can lead edema formation during inflammation (Huang et al., 2011a). Inflammation is initiated through the production of specific cytokines or chemokines characterized by recruitment of leukocytes to the damage site. During inflammation, high levels of reactive oxygen species (ROS) were also produced to exert a defense against pathogens (Chang et al., 2011a). Among them, abnormal excess NO produced by iNOS is believed to act as a toxic radical that can damage cellular macromolecules such as proteins, DNA, and lipids, triggering several unfavorable cellular responses (Huang et al., 2010). For in vivo tests, inflammation can be induced in animals by many substances. Mice paw edema is the most commonly used model for acute inflammation while subcutaneous implantation of biomaterial is usually used for inflammatory model (Chang et al., 2011b). This study was therefore aimed to evaluate the antinociceptive and anti-inflammatory potential of the ethyl acetate fraction of ACE (EA-ACE) in in vivo models. And we detected the levels of iNOS and COX-2 in paw edema. Also, the activities of CAT, SOD, and GPx in the paw tissue at the 5th h after Carr injection were measured to understand the relationship between the anti-inflammatory mechanism of EA-ACE and antioxidant enzymes.

Material and Methods


-Carrageenan (Carr), indomethacin (Indo), and other chemical reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA). TNF-α and IL-1 were purchased from Biosource International Inc., (Camarillo, CA, USA). Anti-iNOS, anti-COX-2, and anti-β-actin antibody (Santa Cruz, USA) and a protein assay kit (Bio-Rad Laboratories Ltd., Watford, Herts, U.K.) were obtained as indicated. Poly-(vinylidene fluoride) membrane (Immobilon-P) was obtained from Millipore Corp. (Bedford, MA, USA). Plant materials were collected from Taichung country in Taiwan. They were identified and authenticated by Dr. Yuan-Shiun Chang, Professor, department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University.

Extraction and fractionation

The coarse powder of the stem of Actinidia callosa var. ephippioides (1 kg) was extracted with methanol three times (10 L). The extract was evaporated under reduced pressure using a rotavapor, and then stored under light protection. A yield equivalent to 5.6 % of the original weight was obtained. Next, methanol extract of ACE (46 g) was dissolved and suspended in 100 mL of water in a separatory funnel prior to being partitioned in sequence with n-hexane, chloroform, ethyl acetate, and n-butanol (800 mL each for three times). Under reduced pressure, fractions were yielded and collected: n-hexane fraction (19.4 g, 42.2%), chloroform fraction (4.9 g, 10.7%), ethyl acetate fraction (12.2 g, 26.5%), n-butanol fraction (5.2 g, 11.3%) and aqueous fraction (4.3 g, 9.3%). All extracts were stored in the refrigerator before the use.

Chemical characterization of EA-ACE extracts by HPLC

HPLC was performed with a Hitachi Liquid Chromatography (Hitachi Ltd., Tokyo, Japan), consisting of two model L-5000 pumps, and one model L-7455 photodiode array detector (254 nm). Samples (10 mg/mL) were filtered through a 0.45 μm PVDF-filter and injected into the HPLC column. The injection volume was 15 μL and the separation temperature was 30°C. The column was a Mightysil RP-18 GP (5 μm, 250 mm × 4.6 mm I.D.). The method involved the use of a binary gradient with mobile phases containing: (A) phosphoric acid in water (0.6, v/v) and (B) MeOH (v/v). The solvent gradient elution program was as follows: from 13% A to 87% A in 20 min. The flow-rate was kept constant at 1.0 mL/min. A precolumn of μ-Bondapak™ C18 (Millipore, Milford, MA, USA) was attached to protect the analytical column. For photodiode array detection, the wavelengths of triterpenic acids at their respective maximum absorbance wavelength can monitored at the same time. All these three compounds were detected at 215 nm. Identification is based on retention times and on-line spectral data in comparison with authentic standards. Quantification is performed by establishing calibration curves for each compound determined, using the standards.


6-8 weeks male imprinting control region (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, and relative humidity of 55 ± 5 % with 12 h dark-light cycle for at least 2 week before the experiment. They were given food and water ad libitum. All experimental procedures were performed according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. In addition, 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 (18-25 g) were randomly assigned to five groups (n=6) of the animals in acetic acid-induced writhing (1%, 0.l mL/10 g i.p.) and formalin-induced licking (5%, 20 L/per mice i.p.) experiments. These include a pathological model group (received acetic acid or formalin), a positive control (acetic acid or formalin + Indo), and EA-ACE administered groups (acetic acid or formalin+ EA-ACE: 62.5, 125, and 250 mg/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 EA-ACE administered groups (Carr + EA-ACE: 62.5, 125, and 250 mg/Kg).
Measurement of Nitric oxide/Nitrite

NO production was indirectly assessed by measuring the nitrite levels in the serum determined by a colorimetric method based on the Griess reaction (Huang et al., 2011b). Serum samples were diluted four times with distilled water and deproteinized by adding 1/20 volume of zinc sulfate (300 g/L) to a final concentration of 15 g/L. After centrifugation at 10,000×g for 5 min at room temperature, 100 μL supernatant was applied to a microtiter plate well, followed by 100 μL of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 5% phosphoric acid). After 10 min of color development at room temperature, the absorbance was measured at 540 nm with a Micro-Reader (Molecular Devices, Orleans Drive, Sunnyvale, CA). By using sodium nitrite to generate a standard curve, the concentration of nitrite was measured by absorbance at 540 nm.

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 EA-ACE-treated groups. Control group received 1% acetic acid (10 mL/Kg body weight) and the positive control group received Indo (10 mg/Kg, p.o.) 25 min before intraperitoneal injection of 1% acetic acid (10 mL/Kg body weight). EA-ACE-treated groups received EA-ACE (62.5, 125, and 250 mg/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 recorded (Chang et al., 2011c).

Formalin test

The antinociceptive activity of the drugs was determined using the formalin test (Chang et al., 2011c). 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 EA-ACE (62.5, 125, and 250 mg/Kg, p.o.) and 30 min after administration of Indo (10 mg/Kg, p.o.). 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 was 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.

Carr-induced Edema

The Carr-induced hind paw edema model was used for determination of anti-inflammatory activity (Wen et al., 2011). Animals were i.p. treated with EA-ACE (62.5, 125, and 250 mg/kg), Indo or normal saline, 30 min prior to injection of 1% Carr (50 μL) in the plantar side of right hind paws of the mice. The paw volume was measured after Carr injection and at 1, 2, 3, 4, and 5 h intervals after the administration of the edematogenic 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 was the volume of the right hind paw before Carr treatment. Indo was used as a positive control. After 5 h, the animals were sacrificed and the Carr-induced edema feet were dissected and stored at -80 ºC. Also, blood were withdrawn and kept at -80 ºC. The protein concentration of the sample was determined by the Bradford dye-binding assay (Bio-Rad, Hercules, CA).

MDA Assay

MDA from Carr-induced edema foot was evaluated by the thiobarbituric acid reacting substance (TRARS) method (Huang et al., 2011c). Briefly, MDA reacted with thiobarbituric acid in the acidic high temperature and formed a red-complex TBARS. The absorbance of TBARS was determined at 532 nm.

Measurement of TNF-α and IL-1 by an Enzyme-Linked Immunosorbent Assay (ELISA)

The levels of TNF- and IL-1 were determined by using a commercially available ELISA kit (Biosource International Inc., Camarillo, CA) according to the manufacturer’s instruction. TNF- and IL-1 were determined from a standard curve.

Antioxidant Enzyme Activity Measurements

The following biochemical parameters were analyzed to check the paw tissues activity of EA-ACE by the methods given below.

Total SOD activity was determined by the inhibition of cytochrome c reduction (Flohe and Otting, 1984). The reduction of cytochrome c was mediated by superoxide anions generated by the xanthine/xanthine oxidase system and monitored at 550 nm. One unit of SOD was defined as the amount of enzyme required to inhibit the rate of cytochrome c reduction by 50%. Total CAT activity was based on that of Aebi (Aebi, 1984). In brief, the reduction of 10mM H2O2 in 20 mM of phosphate buffer (pH 7.0) was monitored by measuring the absorbance at 240 nm. The activity was calculated using a molar absorption coefficient, and the enzyme activities were defined as nanomoles of dissipating hydrogen peroxide per milligram protein per minute. Total GPx activity in cytosol was determined according to Paglia and Valentine’s method (Paglia and Valentine, 1967). 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 activities were defined as nanomoles of NADPH oxidized per milligram protein per minute.
Protein Lysate Preparation and Western blot Analysis of iNOS and COX-2

Soft tissues were removed from individual mice paws and homogenized in a solution containing 10 mM CHAPS, 1 mM phenylmethylsulphonyl fluoride (PMSF), 5 g/mL, aprotinin, 1 M pepstatin and 10 M leupeptin. The homogenates were centrifuged at 12,000g for 20 min, and 30 g of protein from the supernatants was then separated on 10% sodium dodecylsulphate–polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. After transfer, the membrane was blocked for 2 h at room temperature with 5% skim milk in Tris-buffered saline-Tween (TBST; 20 mM Tris, 500 mM NaCl, pH 7.5, 0.1% Tween 20). The membranes were then incubated with mouse monoclonal anti-iNOS, or anti-COX-2 antibody in 5% skim milk in TBST for 2 h at room temperature. The membranes were washed three times with TBST at room temperature and then incubated with a 1 : 2000 dilution of anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (Sigma, St Louis, MO, U.S.A.) in 2.5% skim milk in TBST for 1 h at room temperature. 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 by using Kodak Molecular Imaging Software (Version 4.0.5, Eastman Kodak Company, Rochester, NY) and represented in the relative intensities.

Histological Examination

For histological examination, biopsies of paws took 5 h following the interplanetary injection of Carr. The tissue slices were fixed in a solution (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 hematoxylin and eosin (H&E) stain. All samples were observed and photographed with Nikon microscopy. Every 3-5 tissue slices were randomly chosen from Carr, Indo and EA-ACE-treated (250 mg/kg) groups. Histological examination of these tissue slices revealed an excessive inflammatory response with massive infiltration of neutrophils [ploymorphonuclear leukocytes (PMNs)] by microscopy. The numbers of neutrophils were counted in each scope (400 x) and thereafter we obtained their average count from 5 scopes of every tissue slice.

Statistical Analysis

Data obtained from animal experiments were expressed as mean standard error (± S.E.M.). Statistical evaluation was carried out by one-way analysis of variance (ANOVA followed by Scheffe's multiple range tests). Statistical significance is expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.


Chemical characterization of EA-ACE

To establish the fingerprint chromatogram for the chemical characterization and quality control of EA-ACE. Betulinic acid, ursolic acid, and oleanolic acid were used as markers. An optimized HPLC-PDA technique was employed Meanwhile, HPLC chromatograms showed three marker components present in EA-ACE. As shown in Fig. 1A and 1B, these triperpenic acids have been identified as betulinic acid (retention time, 15.16 min), ursolic acid (17.43 min), and oleanolic acid (18.42 min) by their retention time and UV absorbance of purified standards. According to the plot of peak-area ratio (y) vs. concentration (x, g/mL), the regression equations of the three constituents and their correlation coefficients (r) were determined as follows: betulinic acid, y = 1.085x + 11.383 (r2=0.996); ursolic acid, y = 0.7708x + 6.1583 (r2 = 0.998); and oleanolic acid, y = 1.1074x + 7.7163 (r2 = 0.998). The relative amounts of the three triperpenic acids found in EA-ACE was in the order of oleanolic acid (4.66 mg/g) > ursolic acid (4.50 mg/g) > betulinic acid (0.67 mg/g), respectively.

Acetic acid-induced writhing response

The cumulative amount of abdominal stretching correlated with the level of acetic acid-induced pain (Fig. 2A). EA-ACE treatment (0, 62.5, 125, and 250 mg/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 19.62%, 37.77%, and 50.24% respectively. EA-ACE (250 mg/kg) and positive control Indo (10 mg/kg) significantly inhibited the numbers of acetic acid-induced writhing response (p < 0.001).

Formalin test

The results of formalin test have been summarized in Fig. 2B. EA-ACE significantly inhibited formalin-induced pain in the late phase; however, there was no inhibition in the early phase (Fig. 2B). EA-ACE treatment (0, 62.5, 125, and 250 mg/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 25.22%, 42.56%, and 54.94%, respectively. This inhibiting effect of formalin-induced licking time by EA-ACE (250 mg/kg; p < 0.001) was better than a positive control Indo (10 mg/kg) (p < 0.001).
EA-ACE alleviated Carr-induced mouse paw edema

To determine whether anti-inflammatory effects of EA-ACE occurred in vivo, Carr-induced mouse hind paw edema test was conducted.There was a gradual increase in edema paw volume of mice in the Carr-treated group. However, in the test groups, EA-ACE (250 mg/kg) significantly inhibited Carr-induced mouse paw edemas in a dose-dependent manner with a maximum attend (Fig. 3A). The dose-related inhibition of hind paws edema between 3th to 5th h was observed. Indo as positive control (10 mg/kg) produced a significant inhibitory effect compared to Carr-treated group.

EA-ACE reduced MDA, NO, TNF-and IL-1 productions in vivo

MDA level increased significantly in the edema paw at the 5th h after Carr injection (p < 0.001). As expected, administration of 10 mg/kg Indo significantly reduced the MDA level in the edema paw. In this time, MDA level was also decreased dose-dependently by treatment with EA-ACE (250 mg/kg) (p < 0.001) (Fig. 3B).

In Fig. 3C, the NO level increased significantly in the edema serum after 5th h carrageenan injection (p < 0.001). EA-ACE (125 and 250 mg/kg) markedly decreased the serum NO level (p < 0.01 or p < 0.001) in Carr-treated mice. The inhibitory potency was similar to that of Indo (10 mg/kg) at the 5th h after induction (p < 0.001).

Data from ELISA assay showed that TNF-α and IL-1 levels increased significantly in serum after 5th h Carr injection (p < 0.001). However, EA-ACE (250 mg/kg) and Indo (10 mg/kg) decreased the TNF-α and IL-1 levels in serum at the 5th h after Carr injection (p < 0.001) (Fig. 3D and 3E).

Effects of EA-ACE on activities of antioxidant enzymes

At the 5th h following Carr injection, paw edema tissues were analyzed for the biochemical parameters such as CAT, SOD, and GPx activities (Table 1). CAT, SOD, and GPx activities in paw edema tissue were decreased significantly by Carr administration. CAT, SOD and GPx activity were increased significantly after treated with EA-ACE and 10 mg/kg Indo (P<0.01) (Table 1).

Effects of EA-ACE on Carr-Induced iNOS and COX-2 Protein Expressions in Mice Paw Edema

To investigate whether the inhibition of NO production was due to a decreased iNOS and COX-2 protein level, the effect of EA-ACE on iNOS and COX-2 proteins expression were studied by Western blot. The results showed that injection of EA-ACE (250 mg/kg) on Carr-induced for 5th h inhibited iNOS and COX-2 proteins expression in mouse paw edema (Fig. 4A). The intensity of protein bands were analyzed by using Kodak Quantity software in these three independent experiments and the result showed an average of 57.6% and 56.2% down-regulation of iNOS and COX-2 protein, respectively, after treatment with EA-ACE compared with the Carr-induced alone (Fig. 4B). In addition, the protein expression showed an average of 53.5% and 61.1% down-regulation of iNOS and COX-2 protein after treatment with Indo at 10.0 mg/kg compared with the Carr-induced alone.

Histological examination

Paw biopsies of control animals showed marked cellular infiltration in the connective tissue. The infiltrates accumulated between collagen fibers and into intercellular spaces. Paw biopsies of animals treated with EA-ACE (250 mg/kg) showed a reduction in Carr -induced inflammatory response. Actually inflammatory cells were reduced in numbers and were confined to be 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 hypodermal connective tissue was not damaged (Fig. 5A). The number of neutrophil was significantly increased upon Carr treatment (P < 0.001). However, Indo and EA-ACE (250 mg/kg) could effectively decrease the neutrophil numbers as compared to the Carr-treated group (P < 0.001) (Fig. 5B).


In the present study, we demonstrated anti-analgesic and anti-inflammatory activities of EA-ACE in in vivo experimental systems, using a mouse model of topical inflammation. The inhibitory activities by Carr-induced paw edema in mouse against iNOS, COX-2, TNF-, and IL-1 as shown in efficacy of EA-ACE suggested its potential therapeutic usage as a novel topical anti-inflammatory source of health food.

The acetic writhing test is used to study the peripheral analgesic effects of drugs (Liu et al., 2007). Related studies have demonstrated that acetic acid acts indirectly by inducing the release of endogenous mediators which stimulate the nociceptive neurons sensitive to non-steroidal anti-inflammatory drugs (NSAIDs) (Lin and Shieh, 1996). When compared antinociceptive activities, EA-ACE was relatively potent in acetic acid writhing test indicating peripheral antinociception. In contrast, EA-ACE (250 mg/Kg) exhibited an action in similar magnitude with Indo, a reference drug for peripheral antinociception

The formalin test is a sensitive method for screening various anti-inflammatory and an analgesic drug, which involves neurogenic response and inflammatory action (Lai et al., 2009). The formalin test consists of two-time phases and some previous studies demonstrated that substance P and bradykinin participate in the first phase, whereas histamine, serotonin, PGs, nitrite, and bradykinin were involved in the second phase of the formalin test. The second phase is an inflammatory response with inflammatory pain that can be inhibited by anti-inflammatory drugs (Huang et al., 2012). Therefore, the test can be used to clarify the possible mechanism of an antinociceptive effect of a proposed analgesic. The inhibitory effect of EA-ACE on the nociceptive response in the late phase of the formalin test suggested that the anti-nociceptive effect of EA-ACE could be due to its peripheral action.

In our experiments, intrapleural injection of Carr induced an acute inflammatory reaction, characterized by marked accumulation of a volume of pleural exudates, plasma exudation and intense migration of PMNs in the pleural cavities. Carr-induced edema has been described as a biphasic event. The early phase, observed about 1 h after Carr injection, is related to the production of serotonin, histamine, leukotrienes, bradykinin, and cyclooxygenase products in the inflamed tissue, while the late phase (2–5 h) is due to neutrophil infiltration, as well as to the continuing production of arachidonic acid metabolites (Huang et al., 1998). In a number of pathophysiological conditions associated with inflammation or oxidant stress, these reactive oxygen species have been proposed to mediate cell damage via a number of independent mechanisms including the initiation of lipid peroxidation, the inactivation of a variety of antioxidant enzymes (Achoui et al., 2010). In this results that EA-ACE and Indo significantly inhibited the development of edema 4th h and 5th h after treatment. It is well known that the 3th h of the Carr-induced edema, where the edema reaches its highest volume, is characterized by the injection of Carr into the mice 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.

The l-arginine–NO pathway has been proposed to play an important role in the carrageenan-induced inflammatory response. The expression of the iNOS has been proposed as an important mediator of inflammation (Huang et al., 2012a). In our study, the level of NO was decreased significantly by treatment with 250 mg/kg EA-ACE. We suggest the anti-inflammatory mechanism of EA-ACE may be through the L-arginine–NO pathway because EA-ACE significantly inhibits the NO production.

Pro-inflammatory genes are activated via signal transduction pathways that lead to the production of proinflammatory parameters such as NO, TNF-α, and IL-1β (Huang et al., 2012b). TNF-and IL-1 also are the mediators of Carr-induced inflammatory incapacitation, and are able to induce the further release of kinins and leukotrienes, which are suggested to have an important role in the maintenance of long-lasting nociceptive response (Vasconcelos et al., 2006). In this study, we found that EA-ACE decreased the TNF-α and IL-1 levels in serum after Carr injection.

The Carr-induced inflammatory response could be associated with free radical, such as hydrogen peroxide, superoxide and hydroxyl radicals. MDA production is due to free radical attack on plasma membrane. Thus, inflammatory effect would result in the accumulation of MDA. Glutathione (GSH) acts as a oxyradical scavenger by scavenging NO and other oxidants. The increased GSH level may favor to reduce MDA production. GSH plays an important role against Carr-induced local inflammation (Huang et al., 2011). In this study, there is significantly increased in CAT, SOD, and GPx activities with EA-ACE treatment. Furthermore, there are significantly decreases in MDA level with EA-ACE treatment. We assume the suppression of MDA production is probably due to the increases of CAT, SOD, and GPx activities in the paw edema.

Carr-induced paw edema is a good acute inflammatory model characterized by protein-rich fluid accumulation and PMNs infiltration. This model is a well characterized experimental model of inflammation that permits the assessment of the anti-inflammatory effects of pharmaceutical agents (Huang et al., 2011c). In this study, we demonstrate that EA-ACE can effectively decrease neutrophil migration to sites of acute inflammation.

Effects of betulinic acid, ursolic acid, and oleanolic acid on Carr-induced edema in rats and mice have also been described (Tsai et al., 2011). The inhibitory effects of a number of triterpenes in Carr-induced paw edema tests could be blocked by progesterone, actimomycin D and cycloheximide thus suggesting that the anti-inflammatory actions of these pentacyclic triterpenes are mediated by mechanisms related to glucocorticoid receptor activation and protein biosynthesis (Safayhi and Sailer, 1997). Triterpenoids, all derivatives of oleanolic and ursolic acids, used as potential anti-inflammatory and chemopreventive agents (Singh et al., 1991). These triterpenoids have been tested for their ability to suppress iNOS and COX-2 expressions. The triterpenoid skeleton has no influence on the anti-inflammatory activity and the presence of a carboxylic group at C-28 (C-27 in the lupane series) or C-30 results in an increased activity (Suh et al., 1998). These results suggest that the anti-inflammatory activities of EA-ACE are related to their triterpenic acids.

These results suggested that EA-ACE possessed anti-inflammatory effects. The anti-inflammatory mechanism of EA-ACE may be related to iNOS and it is associated with the increase in the activities of antioxidant enzymes (CAT, SOD, and GPx). EA-ACE may be used as a pharmacological agent in the prevention or treatment of disease in which free radical formation is a pathogenic factor.

The authors want to thank China Medical University (CMU) (CMU100-TC-22, CMU101-SR-54, and CMU-101-AWARD-08) and Taiwan Department of Heath Clinical Trial and Research Center of Excellence (DOH101-TD-B-111-004).


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Table 1: Effects of EA-ACE and indomethacin (Indo) on changes in CAT, SOD and GPx activities were studied on Carr-induced mice paw edema (5th hr).



(U/mg protein)


(U/mg protein)


(U/mg protein)


5.85 ± 0.07

24.27 ± 0.14

23.62± 0.12


3.52 ± 0.01###

16.21 ± 0.03###

15.72 ± 0.07###

Carr+ Indo

5.25 ± 0.05**

23.47 ± 0.09**

22.29 ± 0.08**

Carr + EA-ACE

(62.5 mg/Kg)

3.96 ± 0.12

18.63 ± 0.02

17.38 ± 0.06

Carr + EA-ACE

(125 mg/Kg)

4.56 ± 0.15*

20.57 ± 0.11*

19.61 ± 0.09*

Carr + EA-ACE

(250 mg/Kg)

5.16 ± 0.04**

23.12 ± 0.05**

22.85 ± 0.11**

Each value represents as mean ± S.E.M. ###p < 0.001 as compared with the control group. * p < 0.05 and ** p < 0.01 as compared with the Carr (-carrageenan) group (one-way ANOVA followed by Scheffe’s multiple range test).

Figure Legends

Figure 1. HPLC chromatograms of ethyl-acetate fraction from the stem of Actinidia callosa var. ephippioides (EA-ACE). Standard (A), ethyl-acetate fraction from the stem of Actinidia callosa var. ephippioides (EA-ACE) (B), and the chemical structures of betulinic acid, ursolic acid, and oleanolic acid (B). 1. Betulinic acid; 2. Ursolic acid; and 3. Oleanolic acid.

Figure 2. Analgesic effects of EA-ACE and indomethacin (Indo) were studied on acetic acid-induced writhing response (A) and on the early phase and late phase in formalin test (B) in mice. The values were averaged and obtained in individual animals (n=6). Each value represented as mean ± S.E.M. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the pathological model group (Control) (one-way ANOVA followed by Scheffe’s multiple range test).
Figure 3. Anti-inflammation effect of EA-ACE and Indo on hind paw edema induced by Carr in mice (A), the tissue MDA concentration of foot in mice (B), Carr-induced NO (C), and TNF- (D) concentrations of serum at 5th h in mice. Each value represents as mean ± S.E.M. ###p < 0.001 as compared with the control group. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with the Carr group (one-way ANOVA followed by Scheffe’s multiple range test).
Figure 4. Inhibition of iNOS and COX-2 protein expressions by EA-ACE induced by -carrageenan of foot at 5th h in mice. Tissue suspended were then prepared and subjected to western blotting using an antibody specific for iNOS and COX-2. β-actin was used as an internal control. (A) A representative western blot from two separate experiments is shown. (B) Relative iNOS and COX-2 protein levels were calculated with reference to a Carr-injected mouse. ###compared with sample of control group. The data were presented as mean ± S.D. for three different experiments performed in triplicate. **p < 0.01 and ***p < 0.001 were compared with Carr-alone group.
Figure 5. Representative light micrographs of mouse hind footpad H&E stained to reveal hemorrhage, edema and inflammatory cell infiltration in control mice (A), λ-carrageenan (Carr)-treated mice demonstrates hemorrhage with moderately extravascular red blood cell and large amount of inflammatory leukocyte mainly neutrophils infiltration in the subdermis interstitial tissue of mice, and mice given indomethacin (Indo) (10 mg/kg) before Carr. EA-ACE significantly show morphological alterations (100×) and the numbers of neutrophils in each scope (400x) (B) compared to subcutaneous injection of Carr only. ###p < 0.001 as compared with the control group. ***p < 0.001 compared with Carr group. Scale bar = 200 μm.
Figure 1.




Figure 2.



Figure 3.






Figure 4.



Figure 5.



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