# Involvement of Heme Oxygenase-1 Participates in Anti-Inflammatory and Analgesic Effects of Aqueous Extract ofHibiscus taiwanensis

Authors: Shu-Ling Liu; Jeng-Shyan Deng; Chuan-Sung Chiu; Wen-Chi Hou; Shyh-Shyun Huang; Wang-Ching Lin; Jung-Chun Liao; Guan-Jhong Huang
Journal: Evidence-Based Complementary and Alternative Medicine
 (2012)

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## Abstract

Anti-inflammatory effects of the aqueous extract ofHibiscus taiwanensis (AHT) were used in lipopolysaccharide (LPS-)stimulated mouse macrophage RAW264.7 cells and carrageenan (Carr-)induced mouse paw edema model. When RAW264.7 macrophages were treated with AHT together with LPS, a concentration-dependent inhibition of nitric oxide (NO), tumor necrosis factor (TNF-α), and prostaglandin E2 (PGE2) levels productions were detected.  Western blotting revealed that AHT blocked protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and elevated heme oxygenase-1 (HO-1), significantly. In the animal test, AHT decreased the paw edema at the 4th and the 5th h after Carr administration, and it increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the paw tissue. We also demonstrated AHT decreased the NO, TNF-α, and PGE2 levels on the serum level at the 5th h after the Carr injection. Western blotting revealed that AHT decreased Carr-induced iNOS, and COX-2, and increased HO-1 expressions at the 5th h in the edema paw. These findings demonstrated that AHT has excellent anti-inflammatory activities in vitro and in vivo and thus it has great potential to be used as a source for natural health products.


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## Body

## 1. Introduction

Inflammation involves multiple events regulated by activated inflammatory or immune cells. At the inflammatory site, an increase in blood vessel wall permeability followed by migration of immune cells can lead edema formation during inflammation [1]. Inflammation leads to the upregulation of a series of enzymes and signaling proteins in affected cells and tissues. Inducible nitric oxide synthase (iNOS), a member of the NOS protein family, catalyzes the formation of nitric oxide (NO) from L-arginine [2]. Excess NO production damages tissues and causes transient inflammation. Inducible NOS (iNOS), involved in cellular overproduction of NO, is particularly important in inflammation [3].Macrophage activation by lipopolysaccharides (LPS), which are derived from gram-negative bacteria cell walls, results in the release of several inflammatory mediators including NO, TNF-α, and interleukin-6 [4]. And in the animal, the inflammation model of a carrageenan (Carr-) induced edema is usually used to assess the contribution of 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, and the release of inflammatory mediators [5]. The resulting inflammation has been shown to be associated with a number of chronic diseases, including, asthma, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, and Alzheimer’s disease and also has a role in various human cancers [6].Heme oxygenases (HOs) are responsible for catalyzing heme into carbon monoxide (CO), biliverdin, and free iron. These catabolic heme products mediate some physiological functions, such as, diminishing the production of pro-inflammatory cytokines [7] and stimulating the production of the anti-inflammatory cytokines [8]. Among HOs, heme oxygenase-1 (HO-1) is believed to beneficially play a cytoprotective role in a variety of pathological models, such as, inflammation. The anti-inflammatory properties of HO-1 are related with the inhibition of adhesion molecule expression and reduction of oxidative stress, while exogenous carbon monoxide decreases the production of inflammatory mediators, such as, cytokines and NO [9].Hibiscus taiwanensisHU (Malvaceae) is native to Taiwan. The stem of H. taiwanensis (HT) has been used as anti-inflammatory, antifungal, antipyretic, and anthelmintic agents in the traditional Chinese medicine. A literature review indicated that this genus is a source of lignanamides, naphthalenes, polyphenol compounds, carotenoids, anthocyanins, sterols, and long chain fatty esters [10]. The compounds isolated from HT showed cytotoxic activity against human lung carcinoma and breast carcinoma cell lines [11]. However, in this paper we examined the anti-inflammatory activities of AHT on LPS-induced in RAW264.7 cells and Carr-induced on paw edema in mice. We detected the levels of iNOS, COX-2, and HO-1 in either RAW264.7 cell or 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 AHT and antioxidant enzymes.


## 2. Materials and Methods

### 2.1. Chemicals

LPS (endotoxin fromEscherichia coli, serotype 0127:B8), Carr (Type IV), indomethacin, and other chemicals were purchased from Sigma Chemical Co. (St. Louis, USA). TNF-α and PGE2 were purchased from Biosource International Inc. (Camarillo, CA, USA). Anti-iNOS, anti-COX-2, anti-HO-1, 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 of Taiwan. They were identified and authenticated by Dr. Shyh-Shyun Huang, Assistance professor, Department of Pharmacy, China Medical University, Taichung, Taiwan.


### 2.2. Preparation of the Aqueous Extracts ofHibiscus taiwanensis

A 100 g sample of HT was extracted with water (1 L) at 100°C for 60 min and then centrifuged at  10,000×g for 20 min. The extraction was repeated three times. The extracts were then combined and filtered through a No. 1 filer paper. The filtrates were collected, concentrated with a vacuum evaporator until the volume was below 10 mL, and then freeze dried. The yield obtained was 5.6% (w/w).


### 2.3. Fingerprint Chromatogram of AHT 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 0.45μm PVDF filter and injected into the HPLC column. The injection volume was 10 μL, and the separation temperature was 40°C. The column was a Mightysil RP-18 GP (5 μm, 250mm×4.6mm 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 88% A to 78% A in 30 min, from 78% A to 68% A in 15 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 phenolic compounds at their respective maximum absorbance wavelength can monitored at the same time. 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 by using the standards.


### 2.4. Animals

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 of22±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 μL/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 AHT-administered groups (acetic acid or formalin + AHT: 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 AHT-administered groups (Carr + AHT: 0.25, 0.5, and 1.0 g/Kg).


### 2.5. Cell Culture

A murine macrophage cell line RAW264.7 (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 and 0.02% EDTA in Ca2+-, Mg2+-free phosphate-buffered saline (DPBS).


### 2.6. Cell Viability

Cells (2×105) were cultured in 96-well plate containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. Then cells were cultured with AHT in the presence of 100 ng/mL LPS for 24 h. After that, the cells were washed twice with DPBS and incubated with 100 μL of 0.5 mg/mL MTT for 2 h at 37°C testing for cell viability {MTT, (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide)}. The medium was then discarded and 100 μL dimethyl sulfoxide (DMSO) was added. After the 30 min incubation, absorbance at 570 nm was read by using a microplate reader.


### 2.7. Measurement of Nitric Oxide/Nitrite

NO production was indirectly assessed by measuring the nitrite levels in the cultured media and serum determined by a colorimetric method based on the Griess reaction [2]. The cells were incubated with AHT (0, 125, 250, 500, and 1000 μg/mL) in the presence of LPS (100 ng/mL) at 37°C for 24 h. Then, cells were dispensed into 96-well plates, and 100 mL 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, the absorbance was measured at 540 nm with a Micro-Reader (Molecular Devices, Orleans Drive, Sunnyvale, CA). 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. After 10 min of color development at room temperature, the absorbance was measured at 540 nm with a Micro-Reader. By using sodium nitrite to generate a standard curve, the concentration of nitrite was measured by absorbance at 540 nm.


### 2.8. Evaluation of Acute Toxicity

A single dose of test substance (2.5 and 5.0 g/kg bw) was gavaged to 10 mice in 0.5 mL. Water, providedad libitum, was used as the vehicle in order to estimate the acute toxicity. The animals were monitored for the signs of morbidity and mortality immediately after dosing, at 4 and 24 h, and twice daily for the subsequent 14 days. The animals were sacrificed with CO2 after 14 days following an overnight fast, and a thorough autopsy was performed on all animals. Food and water were provided throughout the experiment. If the mice died, they would be checked for autopsy and biochemical profiles.


### 2.9. Acetic-Acid-Induced Writhing Response

After a 2-week adaptation period, male ICR mice (18–25 g) were randomly assigned to six groups (n=8) including a normal control, an Indo-positive control and four AHT-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). AHT-treated groups received AHT (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 recorded [1].


### 2.10. Formalin Test

The antinociceptive activity of the drugs was determined using the formalin test [1]. Control group received 5% formalin. Twenty microliter of 5% formalin was injected into the dorsal surface of the right hind paw 60 min after administration of AHT (0.25, 0.5, and 1.0 g/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 postformalin injection was referred to as the early phase and the period between 15 min and 40 min as the late phase. The total time that took licking or biting of the injured paw (pain behavior) was measured with a stop watch. The activity was recorded in 5 min intervals.


### 2.11. Carr-Induced Edema

The Carr-induced hind paw edema model was used for determination of anti-inflammatory activity [2]. Animals were i.p. treated with AHT (0.25, 0.5, and 1.0 g/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°C. Also, blood was withdrawn and kept at −80°C. The protein concentration of the sample was determined by the Bradford dye-binding assay (Bio-Rad, Hercules, CA, USA).


### 2.12. MDA Assay

MDA from Carr-induced edema foot was evaluated by the thiobarbituric acid reacting substance (TRARS) method [3]. 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.


### 2.13. Measurement of TNF-α and PGE2 by an Enzyme-Linked Immunosorbent Assay (ELISA)

The levels of TNF- and PGE2 were determined by using a commercially available ELISA kit (Biosource International Inc., Camarillo, CA, USA) according to the manufacturer’s instruction. TNF-α and PGE2 were determined from a standard curve.


### 2.14. Antioxidant Enzyme Activity Measurements

The following biochemical parameters were analyzed to check the paw tissues activity of AHT by the methods given below.Total SOD activity was determined by the inhibition of cytochromec reduction [12]. 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 [13]. In brief, the reduction of 10 mM 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 [14]. 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.


### 2.15. Protein Lysate Preparation and Western Blot Analysis of iNOS, COX-2, and HO-1

The stimulated murine macrophage cell line RAW264.7 cells were washed with PBS and lysed in an ice-cold lysis buffer (10% glycerol, 1% triton X-100, 1 mM Na3VO4, 1 mM EGTA, 10 mM NaF, 1 mM Na4P2O7, 20 mM tris buffer (pH 7.9), 100 mM β-glycerophosphate, 137 mM NaCl, 5 mM EDTA, and one protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA)) on ice for 1 h, followed by centrifugation at 12,000 g for 30 min at 4°C. Soft tissues were removed from individual mice paws and homogenized in a solution containing 10 mM CHAPS, 1 mM phenylmethylsulphonyl fluoride (PMSF), 5 μg/mL, aprotinin, 1 μM pepstatin, and 10 μM leupeptin. The homogenates were centrifuged at 12,000 g for 20 min, and 30 μg 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, anti-COX-2, or anti-HO-1 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, USA) 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, UK). 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, USA) and represented in the relative intensities.


### 2.16. 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 and 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 hematoxylin and eosin (H&E) stain. All samples were observed and photographed with BH-2 Olympus microscopy. Every 3~5 tissue slices were randomly chosen from Carr, Indo, and AHT-treated (1.0 g/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 (400x) and thereafter we obtained their average count from 5 scopes of every tissue slice [15].


### 2.17. Statistical Analysis

Experimental results were presented as the mean± standard deviation (SD) of three parallel measurements. IC50 values were estimated using a nonlinear regression algorithm(SigmaPlot8.0; SPSS Inc. Chicago, IL, USA). 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.


## 2.1. Chemicals

LPS (endotoxin fromEscherichia coli, serotype 0127:B8), Carr (Type IV), indomethacin, and other chemicals were purchased from Sigma Chemical Co. (St. Louis, USA). TNF-α and PGE2 were purchased from Biosource International Inc. (Camarillo, CA, USA). Anti-iNOS, anti-COX-2, anti-HO-1, 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 of Taiwan. They were identified and authenticated by Dr. Shyh-Shyun Huang, Assistance professor, Department of Pharmacy, China Medical University, Taichung, Taiwan.


## 2.2. Preparation of the Aqueous Extracts ofHibiscus taiwanensis

A 100 g sample of HT was extracted with water (1 L) at 100°C for 60 min and then centrifuged at  10,000×g for 20 min. The extraction was repeated three times. The extracts were then combined and filtered through a No. 1 filer paper. The filtrates were collected, concentrated with a vacuum evaporator until the volume was below 10 mL, and then freeze dried. The yield obtained was 5.6% (w/w).


## 2.3. Fingerprint Chromatogram of AHT 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 0.45μm PVDF filter and injected into the HPLC column. The injection volume was 10 μL, and the separation temperature was 40°C. The column was a Mightysil RP-18 GP (5 μm, 250mm×4.6mm 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 88% A to 78% A in 30 min, from 78% A to 68% A in 15 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 phenolic compounds at their respective maximum absorbance wavelength can monitored at the same time. 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 by using the standards.


## 2.4. Animals

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 of22±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 μL/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 AHT-administered groups (acetic acid or formalin + AHT: 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 AHT-administered groups (Carr + AHT: 0.25, 0.5, and 1.0 g/Kg).


## 2.5. Cell Culture

A murine macrophage cell line RAW264.7 (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 and 0.02% EDTA in Ca2+-, Mg2+-free phosphate-buffered saline (DPBS).


## 2.6. Cell Viability

Cells (2×105) were cultured in 96-well plate containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. Then cells were cultured with AHT in the presence of 100 ng/mL LPS for 24 h. After that, the cells were washed twice with DPBS and incubated with 100 μL of 0.5 mg/mL MTT for 2 h at 37°C testing for cell viability {MTT, (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide)}. The medium was then discarded and 100 μL dimethyl sulfoxide (DMSO) was added. After the 30 min incubation, absorbance at 570 nm was read by using a microplate reader.


## 2.7. Measurement of Nitric Oxide/Nitrite

NO production was indirectly assessed by measuring the nitrite levels in the cultured media and serum determined by a colorimetric method based on the Griess reaction [2]. The cells were incubated with AHT (0, 125, 250, 500, and 1000 μg/mL) in the presence of LPS (100 ng/mL) at 37°C for 24 h. Then, cells were dispensed into 96-well plates, and 100 mL 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, the absorbance was measured at 540 nm with a Micro-Reader (Molecular Devices, Orleans Drive, Sunnyvale, CA). 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. After 10 min of color development at room temperature, the absorbance was measured at 540 nm with a Micro-Reader. By using sodium nitrite to generate a standard curve, the concentration of nitrite was measured by absorbance at 540 nm.


## 2.8. Evaluation of Acute Toxicity

A single dose of test substance (2.5 and 5.0 g/kg bw) was gavaged to 10 mice in 0.5 mL. Water, providedad libitum, was used as the vehicle in order to estimate the acute toxicity. The animals were monitored for the signs of morbidity and mortality immediately after dosing, at 4 and 24 h, and twice daily for the subsequent 14 days. The animals were sacrificed with CO2 after 14 days following an overnight fast, and a thorough autopsy was performed on all animals. Food and water were provided throughout the experiment. If the mice died, they would be checked for autopsy and biochemical profiles.


## 2.9. Acetic-Acid-Induced Writhing Response

After a 2-week adaptation period, male ICR mice (18–25 g) were randomly assigned to six groups (n=8) including a normal control, an Indo-positive control and four AHT-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). AHT-treated groups received AHT (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 recorded [1].


## 2.10. Formalin Test

The antinociceptive activity of the drugs was determined using the formalin test [1]. Control group received 5% formalin. Twenty microliter of 5% formalin was injected into the dorsal surface of the right hind paw 60 min after administration of AHT (0.25, 0.5, and 1.0 g/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 postformalin injection was referred to as the early phase and the period between 15 min and 40 min as the late phase. The total time that took licking or biting of the injured paw (pain behavior) was measured with a stop watch. The activity was recorded in 5 min intervals.


## 2.11. Carr-Induced Edema

The Carr-induced hind paw edema model was used for determination of anti-inflammatory activity [2]. Animals were i.p. treated with AHT (0.25, 0.5, and 1.0 g/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°C. Also, blood was withdrawn and kept at −80°C. The protein concentration of the sample was determined by the Bradford dye-binding assay (Bio-Rad, Hercules, CA, USA).


## 2.12. MDA Assay

MDA from Carr-induced edema foot was evaluated by the thiobarbituric acid reacting substance (TRARS) method [3]. 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.


## 2.13. Measurement of TNF-α and PGE2 by an Enzyme-Linked Immunosorbent Assay (ELISA)

The levels of TNF- and PGE2 were determined by using a commercially available ELISA kit (Biosource International Inc., Camarillo, CA, USA) according to the manufacturer’s instruction. TNF-α and PGE2 were determined from a standard curve.


## 2.14. Antioxidant Enzyme Activity Measurements

The following biochemical parameters were analyzed to check the paw tissues activity of AHT by the methods given below.Total SOD activity was determined by the inhibition of cytochromec reduction [12]. 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 [13]. In brief, the reduction of 10 mM 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 [14]. 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.


## 2.15. Protein Lysate Preparation and Western Blot Analysis of iNOS, COX-2, and HO-1

The stimulated murine macrophage cell line RAW264.7 cells were washed with PBS and lysed in an ice-cold lysis buffer (10% glycerol, 1% triton X-100, 1 mM Na3VO4, 1 mM EGTA, 10 mM NaF, 1 mM Na4P2O7, 20 mM tris buffer (pH 7.9), 100 mM β-glycerophosphate, 137 mM NaCl, 5 mM EDTA, and one protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA)) on ice for 1 h, followed by centrifugation at 12,000 g for 30 min at 4°C. Soft tissues were removed from individual mice paws and homogenized in a solution containing 10 mM CHAPS, 1 mM phenylmethylsulphonyl fluoride (PMSF), 5 μg/mL, aprotinin, 1 μM pepstatin, and 10 μM leupeptin. The homogenates were centrifuged at 12,000 g for 20 min, and 30 μg 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, anti-COX-2, or anti-HO-1 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, USA) 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, UK). 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, USA) and represented in the relative intensities.


## 2.16. 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 and 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 hematoxylin and eosin (H&E) stain. All samples were observed and photographed with BH-2 Olympus microscopy. Every 3~5 tissue slices were randomly chosen from Carr, Indo, and AHT-treated (1.0 g/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 (400x) and thereafter we obtained their average count from 5 scopes of every tissue slice [15].


## 2.17. Statistical Analysis

Experimental results were presented as the mean± standard deviation (SD) of three parallel measurements. IC50 values were estimated using a nonlinear regression algorithm(SigmaPlot8.0; SPSS Inc. Chicago, IL, USA). 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.


## 3. Results

### 3.1. Fingerprint Chromatogram of HPLC

To establish the fingerprint chromatogram for the quality control of AHT, gallic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid,p-coumaric acid, and ferulic acid were used as markers. An optimized HPLC-PDA technique was employed. Meanwhile, HPLC chromatograms showed seven marker components present in AHT. As shown in Figure 1, these phenolic components have been identified as gallic acid (retention time, 5.3 min), chlorogenic acid (17.8 min), vanillic acid (21.3 min), caffeic acid (22.3 min), syringic acid (26.2 min), p-coumaric acid (34.5 min), and ferulic acid (40.6 min) by their retention time and UV absorbance of purified standards. According to the plot of peak-area ratio (y) versus concentration (x, μg/mL), the regression equations of the four constituents and their correlation coefficients (r) were determined as follows: gallic acid, y=1.111x+8.101(r2=0.999); chlorogenic acid, y=75775x-1E+06(r2=0.9984); vanillic acid, y=52766x-899466(r2=0.9976); caffeic acid, y=111501x-1E+06(r2=0.9984), syringic acid, y=y=1.410x+10.19(r2=0.999); p-coumaric acid, y=0.443x+3.104(r2=0.999); ferulic acid, y=1.283x+8.802(r2=0.999). The relative amounts of the seven phenolic compounds found in AHT were in the order of ferulic acid (21.52 mg/g)> p-coumaric acid (18.53 mg/g)> gallic acid (1.65 mg/g)> syringic acid (0.94 mg/g)> chlorogenic acid (0.38 mg/g) > vanillic acid (0.32 mg/g) > and caffeic acid (0.22 mg/g), respectively.Figure 1
HPLC chromatogram of the aqueous extract ofHibiscus taiwanensis (AHT). The peaks indicate the following, (1) gallic acid; (2) chlorogenic acid; (3) vanillic acid; (4) caffeic acid; (5) syringic acid; (6) p-coumaric acid; (7) ferulic acid.


### 3.2. Cell Viability

The effect of AHT on RAW264.7 cell viability was determined by a MTT assay. Cells cultured with samples at the concentrations (0, 62.5, 125, 250, 500, and 1,000μg/mL) used in the presence of 100 ng/mL LPS for 24 h did not change cell viability (Figure 2(a)).Effects of AHT on lipopolysaccharide (LPS-) induced cell viability (a), NO (b), TNF-α (c), PGE2 (d) productions, and iNOS, COX-2, and HO-1 expressions (e). Cells were incubated for 24 hrs with 100 ng/mL of LPS in the absence or presence of AHT (0, 62.5, 125, 250, 500, and 1,000 μg/mL). ARR was added 1 h before incubation with LPS. The data were presented as mean ± S.D. for three different experiments performed in triplicate. ###Compared with sample of control group. *P<0.05, **P<0.01, and ***P<0.001 were compared with LPS-alone group.
(a)
(b)
(c)
(d)
(e)


### 3.3. Effect of AHT on LPS-Induced NO Production in Macrophages

In the present study, the effects of AHT on LPS-induced NO production in RAW264.7 macrophages were investigated. Nitrite accumulated in the culture medium was estimated by the Griess reaction as an index for NO release from the cells. After the treatment with LPS (100 ng/mL) for 24 h, the nitrite concentration increased in the medium. When RAW264.7 macrophages were treated with different concentrations of AHT together with LPS for 24 h, the AHT inhibited nitrite production significantly (Figure2(b)). AHT did not interfere with the reaction between nitrite and Griess reagents at 1,000 μg/mL (data not shown). Unstimulated macrophages after 24 h of incubation in culture medium produced background levels of nitrite. When RAW264.7 macrophages were treated with different concentrations of AHT (0, 62.5, 125, 250, 500, and 1,000 μg/mL) together with LPS (100 ng/mL) for 24 h, a significant concentration-dependent inhibition of nitrite production was detected. There was either a significant decrease in the nitrite production of group treated with 250 μg/mL AHT (P<0.05) or very and highly significant decrease of groups treated, respectively, with 500 and 1,000 μg/mL of AHT when compared with the LPS-alone group (P<0.01 or P<0.001). The IC50 value for inhibition of nitrite production of AHT was about 786.59±5.28 μg/mL.


### 3.4. Inhibition of LPS-Induced the Level of TNF-α and PGE2 by AHT

We examined the effect of AHT on LPS-induced upregulation of TNF-α. A very low amount of TNF-α protein was detected by a specific ELISA for TNF-α in controls (Figure 2(c)). When RAW264.7 macrophages were treated with different concentrations of AHT (0, 62.5, 125, 250, 500, and 1,000 μg/mL) together with LPS (100 ng/mL) for 24 h, a significant concentration-dependent inhibition of TNF-α production was detected. There was either a significant decrease in the TNF-α production of group treated with 125 and 250 μg/mL AHT (P<0.05) or highly significant decrease of groups treated, respectively, with  500, and 1,000 μg/mL of AHT when compared with the LPS-alone group (P<0.01 or P<0.001). The IC50 value for inhibition of TNF-α production of AHT was about 728.89±2.45μg/mL.PGE2 represents the most important inflammatory product of COX-2 activity, and it was quantified in cell-free culture supernatant [16]. As shown in Figure 2(d), cells were stimulated with LPS alone raised significant amount of PGE2 in RAW264.7 macrophages. When RAW264.7 macrophages were treated with different concentrations of AHT (0, 62.5, 125, 250, 500, and 1,000 μg/mL) together with LPS (100 ng/mL) for 24 h, a significant concentration-dependent inhibition of PGE2 production was detected. The IC50 value for inhibition of PGE2 production of AHT was about 704.71±3.58 μg/mL.


### 3.5. Effects of LPS-Induced iNOS, COX-2, and HO-1 Protein by AHT

In order to investigate whether the inhibition of NO production was due to a decreased iNOS and COX-2 protein level, the effect of AHT was studied by immunoblot. The results showed the incubation with AHT (0, 250, 500, and 1,000μg/mL) in the presence of LPS (100 ng/mL) for 24 h inhibited iNOS and COX-2 proteins expression in mouse macrophage RAW264.7 cells in the cytosol (Figure 2(e)). The detection of β-actin was also performed in the same blot as an internal control. The intensity of protein bands was analyzed by using Kodak Quantity software in three independent experiments, and it showed an average of 83.8% and 70.7% downregulation of iNOS and COX-2 proteins, respectively, after treatment with AHT at 1,000 μg/mL compared with the LPS-alone (Figure 2(e)). Also our study evidenced that the intensity of protein bands showed an average of 89.3% upregulation of HO-1 protein (P<0.001) (Figure 2(e)).


### 3.6. Evaluation of Acute Toxicity in Mice

Oral administration of AHT was found to be safe in acute toxicity. For 24 h after oral administration, any abnormal behavior was not observed, and the mortality was noted asLD50>5 g/kg in mice. Five g/kg dose of AHT, which is a safe dose, was used for further animal test.


### 3.7. Acetic-Acid-Induced Writhing Response

The cumulative amount of abdominal stretching correlated with the level of acetic-acid-induced pain (Figure3(a)). AHT 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 25.53%, 49.23%, and 61.71%, respectively. The inhibiting effect of acetic-acid-induced writhing by AHT (1.0 g/kg) was similar to that produced by a positive control Indo (10 mg/kg) (P<0.001).Analgesic effects of AHT and Indo on acetic-acid-induced writhing response (a) and on the early phase and late phase in formalin test (b) in mice. The values are averaged and obtained in individual animals (n=6). Each value represents 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).
(a)
(b)


### 3.8. Formalin Test

AHT significantly inhibited formalin-induced pain in the late phase; however, there was no inhibition in the early phase (Figure3(b)). AHT 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 22.72%, 41.73%, and 54.48%, respectively. This inhibiting effect of formalin-induced licking time by AHT (1.0 g/kg; P<0.001) was better than a positive control Indo (10 mg/kg) (P<0.001).


### 3.9. Effects of AHT on Carr-Induced Mice Paw Edema

In this study, we used Carr-induced edema because this model is widely employed for screening the effects of anti-inflammatory drugs. Carr-induced paw edema was shown in Figure4(a). AHT (1.0 g/kg) inhibited (P<0.001) the development of paw edema induced by Carr after the 4th and the 5th h of treatment, significantly. Indo (10 mg/kg) significantly decreased the Carr-induced paw edema after the 4th and the 5th h of treatment (P<0.001).Effects of AHT 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), TNF-α (d), and PGE2 (e) concentrations of serum at the 5th hr in mice. The values are averaged and obtained in individual animals (n=6). Each value represents as mean ± S.E.M. ###Compared with sample of 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).
(a)
(b)
(c)
(d)
(e)


### 3.10. Effects of AHT on the MDA Level

The MDA level increased significantly in the edema paw at the 5th h after Carr injection (P<0.001). However, the MDA level was decreased significantly by treatment with AHT (1.0 g/kg) (P<0.001), as well as 10 mg/kg Indo (Figure 4(b)).


### 3.11. Effects of AHT on the NO Level

In Figure4(c), the NO level increased significantly in the edema serum at the 5th h after Carr injection (P<0.001). AHT (1.0 g/kg) significantly decreased the serum NO level (P<0.001). The inhibitory potency was similar to that of Indo (10 mg/kg) at the 5th h after induction.


### 3.12. Effects of AHT on the TNF-α and PGE2 Level

TNF-α and PGE2 level increased significantly in serum at the 5th h after Carr injection (P<0.001). However, AHT (1.0 g/kg) decreased the TNF-α and PGE2 level in serum at the 5th h after Carr injection (P<0.001), as well as 10 mg/kg Indo (Figures 4(d) and 4(e)).


### 3.13. Effects of AHT on Activities of Antioxidant Enzymes

At the 5th h after the intrapaw injection of Carr, paw tissues were also analyzed for the biochemical parameters, such as CAT, SOD, and GPx activities. CAT, SOD, and GPx activities in paw tissue were decreased significantly by Carr administration. CAT, SOD, and GPx activities were increased significantly after treated with 1.0 g/kg AHT and 10 mg/kg Indo (P<0.01) (Table 1).Table 1
Effects of AHT and indomethacin (Indo) on changes in CAT, SOD, and GPx activities were studied on Carr-induced mice paw edema (5th h).
Groups
CAT (U/mg protein)
SOD (U/mg protein)
GPx  (U/mg protein)
Control
5.89 ± 0.15
24.87 ± 0.13
23.35 ± 0.16
Carr
2.28 ± 0.08###
14.64 ± 0.25###
13.64 ± 0.11###
Carr + Indo (10 mg/kg)
5.06 ± 0.24**
22.17 ± 0.10**
21.59 ± 0.19**
Carr + AHT (0.25 g/kg)
3.19 ± 0.14
16.24 ± 0.16
15.78 ± 0.13
Carr + AHT (0.5 g/kg)
3.85 ± 0.09*
19.49 ± 0.07*
18.45 ± 0.18*
Carr + AHT (1.0 g/kg)
5.17 ± 0.10**
22.08 ± 0.18**
20.96 ± 0.23**
*The values are averaged and obtained in individual animals (n=6). 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 (λ-carrageenan) group (one-way ANOVA followed by Scheffe’s multiple range test).


### 3.14. Effects of AHT on Carr-Induced iNOS, COX-2, and HO-1 Protein Expressions in Mice Paw Edema

To investigate whether the inhibition of NO production was due to a decreased iNOS, and COX-2 protein levels, the effect of AHT on iNOS and COX-2 proteins expression was studied by western blot. The results showed that injection of AHT (1.0 g/kg) on Carr-induced for 5 h inhibited iNOS and COX-2 proteins expression in mouse paw edema (Figure5(a)). The detection of β-actin was also performed in the same blot as an internal control. The intensity of protein bands was analyzed by using Kodak Quantity software in three independent experiments and the outcome of it showed an average of 67.6% and 72.4.3% downregulation of iNOS and COX-2 protein, respectively, after the treatment with AHT at 1.0 g/kg compared with the Carr-induced alone (Figure 5(b)). In addition, the protein expression showed an average of 63.6% and 51.2% downregulation of iNOS and COX-2 protein after the treatment with Indo at 10 mg/kg compared with the Carr-induced alone. And we also found that the intensity of protein bands showed an average of 82.5% upregulation of HO-1 protein (P<0.001) (Figure 5(b)).The inhibition of iNOS, COX-2, and HO-1 protein expression by AHT induced by Carr of foot at 5th hr in mice. Tissue suspended was 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 was shown. (b) Relative iNOS, COX-2, and HO-1 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<0.001 was compared with the Carr-alone group (one-way ANOVA followed by Scheffe’s multiple range test).
(a)
(b)


### 3.15. Histological Examination

Paw biopsies of Carr model 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 AHT (1.0 g/kg) showed a reduction in Carr-induced inflammatory response. Actually inflammatory cells were reduced in number and 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 (Figure6(a)). Neutrophils increased with Carr treatment (P<0.01). Indo and AHT could significantly decrease the neutrophils numbers as compared to the Carr-treated group (P<0.001) (Figure 6(b)).Histological appearance of the mouse hind footpad after a subcutaneous injection with Carr stained with H&E stain at the 5th hr to reveal hemorrhage, edema, and inflammatory cell infiltration in control mice, Carr-treated mice demonstrating hemorrhage with moderately extravascular red blood cells and a large amount of inflammatory leukocyte mainly neutrophils infiltration in the subdermis interstitial tissue of mice, and mice given Indo (10 mg/kg) before Carr. AHT (1.0 g/kg) significantly shows morphological alterations (100x) (a) and the numbers of neutrophils in each scope (400x) compared to subcutaneous injection of Carr only (b).###P<0.001 as compared with the control group. ***P<0.001 compared with the Carr group. Scale bar = 100 μm (one-way ANOVA followed by Scheffe’s multiple range test).
(a)
(b)


## 3.1. Fingerprint Chromatogram of HPLC

To establish the fingerprint chromatogram for the quality control of AHT, gallic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid,p-coumaric acid, and ferulic acid were used as markers. An optimized HPLC-PDA technique was employed. Meanwhile, HPLC chromatograms showed seven marker components present in AHT. As shown in Figure 1, these phenolic components have been identified as gallic acid (retention time, 5.3 min), chlorogenic acid (17.8 min), vanillic acid (21.3 min), caffeic acid (22.3 min), syringic acid (26.2 min), p-coumaric acid (34.5 min), and ferulic acid (40.6 min) by their retention time and UV absorbance of purified standards. According to the plot of peak-area ratio (y) versus concentration (x, μg/mL), the regression equations of the four constituents and their correlation coefficients (r) were determined as follows: gallic acid, y=1.111x+8.101(r2=0.999); chlorogenic acid, y=75775x-1E+06(r2=0.9984); vanillic acid, y=52766x-899466(r2=0.9976); caffeic acid, y=111501x-1E+06(r2=0.9984), syringic acid, y=y=1.410x+10.19(r2=0.999); p-coumaric acid, y=0.443x+3.104(r2=0.999); ferulic acid, y=1.283x+8.802(r2=0.999). The relative amounts of the seven phenolic compounds found in AHT were in the order of ferulic acid (21.52 mg/g)> p-coumaric acid (18.53 mg/g)> gallic acid (1.65 mg/g)> syringic acid (0.94 mg/g)> chlorogenic acid (0.38 mg/g) > vanillic acid (0.32 mg/g) > and caffeic acid (0.22 mg/g), respectively.Figure 1
HPLC chromatogram of the aqueous extract ofHibiscus taiwanensis (AHT). The peaks indicate the following, (1) gallic acid; (2) chlorogenic acid; (3) vanillic acid; (4) caffeic acid; (5) syringic acid; (6) p-coumaric acid; (7) ferulic acid.


## 3.2. Cell Viability

The effect of AHT on RAW264.7 cell viability was determined by a MTT assay. Cells cultured with samples at the concentrations (0, 62.5, 125, 250, 500, and 1,000μg/mL) used in the presence of 100 ng/mL LPS for 24 h did not change cell viability (Figure 2(a)).Effects of AHT on lipopolysaccharide (LPS-) induced cell viability (a), NO (b), TNF-α (c), PGE2 (d) productions, and iNOS, COX-2, and HO-1 expressions (e). Cells were incubated for 24 hrs with 100 ng/mL of LPS in the absence or presence of AHT (0, 62.5, 125, 250, 500, and 1,000 μg/mL). ARR was added 1 h before incubation with LPS. The data were presented as mean ± S.D. for three different experiments performed in triplicate. ###Compared with sample of control group. *P<0.05, **P<0.01, and ***P<0.001 were compared with LPS-alone group.
(a)
(b)
(c)
(d)
(e)


## 3.3. Effect of AHT on LPS-Induced NO Production in Macrophages

In the present study, the effects of AHT on LPS-induced NO production in RAW264.7 macrophages were investigated. Nitrite accumulated in the culture medium was estimated by the Griess reaction as an index for NO release from the cells. After the treatment with LPS (100 ng/mL) for 24 h, the nitrite concentration increased in the medium. When RAW264.7 macrophages were treated with different concentrations of AHT together with LPS for 24 h, the AHT inhibited nitrite production significantly (Figure2(b)). AHT did not interfere with the reaction between nitrite and Griess reagents at 1,000 μg/mL (data not shown). Unstimulated macrophages after 24 h of incubation in culture medium produced background levels of nitrite. When RAW264.7 macrophages were treated with different concentrations of AHT (0, 62.5, 125, 250, 500, and 1,000 μg/mL) together with LPS (100 ng/mL) for 24 h, a significant concentration-dependent inhibition of nitrite production was detected. There was either a significant decrease in the nitrite production of group treated with 250 μg/mL AHT (P<0.05) or very and highly significant decrease of groups treated, respectively, with 500 and 1,000 μg/mL of AHT when compared with the LPS-alone group (P<0.01 or P<0.001). The IC50 value for inhibition of nitrite production of AHT was about 786.59±5.28 μg/mL.


## 3.4. Inhibition of LPS-Induced the Level of TNF-α and PGE2 by AHT

We examined the effect of AHT on LPS-induced upregulation of TNF-α. A very low amount of TNF-α protein was detected by a specific ELISA for TNF-α in controls (Figure 2(c)). When RAW264.7 macrophages were treated with different concentrations of AHT (0, 62.5, 125, 250, 500, and 1,000 μg/mL) together with LPS (100 ng/mL) for 24 h, a significant concentration-dependent inhibition of TNF-α production was detected. There was either a significant decrease in the TNF-α production of group treated with 125 and 250 μg/mL AHT (P<0.05) or highly significant decrease of groups treated, respectively, with  500, and 1,000 μg/mL of AHT when compared with the LPS-alone group (P<0.01 or P<0.001). The IC50 value for inhibition of TNF-α production of AHT was about 728.89±2.45μg/mL.PGE2 represents the most important inflammatory product of COX-2 activity, and it was quantified in cell-free culture supernatant [16]. As shown in Figure 2(d), cells were stimulated with LPS alone raised significant amount of PGE2 in RAW264.7 macrophages. When RAW264.7 macrophages were treated with different concentrations of AHT (0, 62.5, 125, 250, 500, and 1,000 μg/mL) together with LPS (100 ng/mL) for 24 h, a significant concentration-dependent inhibition of PGE2 production was detected. The IC50 value for inhibition of PGE2 production of AHT was about 704.71±3.58 μg/mL.


## 3.5. Effects of LPS-Induced iNOS, COX-2, and HO-1 Protein by AHT

In order to investigate whether the inhibition of NO production was due to a decreased iNOS and COX-2 protein level, the effect of AHT was studied by immunoblot. The results showed the incubation with AHT (0, 250, 500, and 1,000μg/mL) in the presence of LPS (100 ng/mL) for 24 h inhibited iNOS and COX-2 proteins expression in mouse macrophage RAW264.7 cells in the cytosol (Figure 2(e)). The detection of β-actin was also performed in the same blot as an internal control. The intensity of protein bands was analyzed by using Kodak Quantity software in three independent experiments, and it showed an average of 83.8% and 70.7% downregulation of iNOS and COX-2 proteins, respectively, after treatment with AHT at 1,000 μg/mL compared with the LPS-alone (Figure 2(e)). Also our study evidenced that the intensity of protein bands showed an average of 89.3% upregulation of HO-1 protein (P<0.001) (Figure 2(e)).


## 3.6. Evaluation of Acute Toxicity in Mice

Oral administration of AHT was found to be safe in acute toxicity. For 24 h after oral administration, any abnormal behavior was not observed, and the mortality was noted asLD50>5 g/kg in mice. Five g/kg dose of AHT, which is a safe dose, was used for further animal test.


## 3.7. Acetic-Acid-Induced Writhing Response

The cumulative amount of abdominal stretching correlated with the level of acetic-acid-induced pain (Figure3(a)). AHT 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 25.53%, 49.23%, and 61.71%, respectively. The inhibiting effect of acetic-acid-induced writhing by AHT (1.0 g/kg) was similar to that produced by a positive control Indo (10 mg/kg) (P<0.001).Analgesic effects of AHT and Indo on acetic-acid-induced writhing response (a) and on the early phase and late phase in formalin test (b) in mice. The values are averaged and obtained in individual animals (n=6). Each value represents 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).
(a)
(b)


## 3.8. Formalin Test

AHT significantly inhibited formalin-induced pain in the late phase; however, there was no inhibition in the early phase (Figure3(b)). AHT 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 22.72%, 41.73%, and 54.48%, respectively. This inhibiting effect of formalin-induced licking time by AHT (1.0 g/kg; P<0.001) was better than a positive control Indo (10 mg/kg) (P<0.001).


## 3.9. Effects of AHT on Carr-Induced Mice Paw Edema

In this study, we used Carr-induced edema because this model is widely employed for screening the effects of anti-inflammatory drugs. Carr-induced paw edema was shown in Figure4(a). AHT (1.0 g/kg) inhibited (P<0.001) the development of paw edema induced by Carr after the 4th and the 5th h of treatment, significantly. Indo (10 mg/kg) significantly decreased the Carr-induced paw edema after the 4th and the 5th h of treatment (P<0.001).Effects of AHT 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), TNF-α (d), and PGE2 (e) concentrations of serum at the 5th hr in mice. The values are averaged and obtained in individual animals (n=6). Each value represents as mean ± S.E.M. ###Compared with sample of 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).
(a)
(b)
(c)
(d)
(e)


## 3.10. Effects of AHT on the MDA Level

The MDA level increased significantly in the edema paw at the 5th h after Carr injection (P<0.001). However, the MDA level was decreased significantly by treatment with AHT (1.0 g/kg) (P<0.001), as well as 10 mg/kg Indo (Figure 4(b)).


## 3.11. Effects of AHT on the NO Level

In Figure4(c), the NO level increased significantly in the edema serum at the 5th h after Carr injection (P<0.001). AHT (1.0 g/kg) significantly decreased the serum NO level (P<0.001). The inhibitory potency was similar to that of Indo (10 mg/kg) at the 5th h after induction.


## 3.12. Effects of AHT on the TNF-α and PGE2 Level

TNF-α and PGE2 level increased significantly in serum at the 5th h after Carr injection (P<0.001). However, AHT (1.0 g/kg) decreased the TNF-α and PGE2 level in serum at the 5th h after Carr injection (P<0.001), as well as 10 mg/kg Indo (Figures 4(d) and 4(e)).


## 3.13. Effects of AHT on Activities of Antioxidant Enzymes

At the 5th h after the intrapaw injection of Carr, paw tissues were also analyzed for the biochemical parameters, such as CAT, SOD, and GPx activities. CAT, SOD, and GPx activities in paw tissue were decreased significantly by Carr administration. CAT, SOD, and GPx activities were increased significantly after treated with 1.0 g/kg AHT and 10 mg/kg Indo (P<0.01) (Table 1).Table 1
Effects of AHT and indomethacin (Indo) on changes in CAT, SOD, and GPx activities were studied on Carr-induced mice paw edema (5th h).
Groups
CAT (U/mg protein)
SOD (U/mg protein)
GPx  (U/mg protein)
Control
5.89 ± 0.15
24.87 ± 0.13
23.35 ± 0.16
Carr
2.28 ± 0.08###
14.64 ± 0.25###
13.64 ± 0.11###
Carr + Indo (10 mg/kg)
5.06 ± 0.24**
22.17 ± 0.10**
21.59 ± 0.19**
Carr + AHT (0.25 g/kg)
3.19 ± 0.14
16.24 ± 0.16
15.78 ± 0.13
Carr + AHT (0.5 g/kg)
3.85 ± 0.09*
19.49 ± 0.07*
18.45 ± 0.18*
Carr + AHT (1.0 g/kg)
5.17 ± 0.10**
22.08 ± 0.18**
20.96 ± 0.23**
*The values are averaged and obtained in individual animals (n=6). 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 (λ-carrageenan) group (one-way ANOVA followed by Scheffe’s multiple range test).


## 3.14. Effects of AHT on Carr-Induced iNOS, COX-2, and HO-1 Protein Expressions in Mice Paw Edema

To investigate whether the inhibition of NO production was due to a decreased iNOS, and COX-2 protein levels, the effect of AHT on iNOS and COX-2 proteins expression was studied by western blot. The results showed that injection of AHT (1.0 g/kg) on Carr-induced for 5 h inhibited iNOS and COX-2 proteins expression in mouse paw edema (Figure5(a)). The detection of β-actin was also performed in the same blot as an internal control. The intensity of protein bands was analyzed by using Kodak Quantity software in three independent experiments and the outcome of it showed an average of 67.6% and 72.4.3% downregulation of iNOS and COX-2 protein, respectively, after the treatment with AHT at 1.0 g/kg compared with the Carr-induced alone (Figure 5(b)). In addition, the protein expression showed an average of 63.6% and 51.2% downregulation of iNOS and COX-2 protein after the treatment with Indo at 10 mg/kg compared with the Carr-induced alone. And we also found that the intensity of protein bands showed an average of 82.5% upregulation of HO-1 protein (P<0.001) (Figure 5(b)).The inhibition of iNOS, COX-2, and HO-1 protein expression by AHT induced by Carr of foot at 5th hr in mice. Tissue suspended was 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 was shown. (b) Relative iNOS, COX-2, and HO-1 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<0.001 was compared with the Carr-alone group (one-way ANOVA followed by Scheffe’s multiple range test).
(a)
(b)


## 3.15. Histological Examination

Paw biopsies of Carr model 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 AHT (1.0 g/kg) showed a reduction in Carr-induced inflammatory response. Actually inflammatory cells were reduced in number and 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 (Figure6(a)). Neutrophils increased with Carr treatment (P<0.01). Indo and AHT could significantly decrease the neutrophils numbers as compared to the Carr-treated group (P<0.001) (Figure 6(b)).Histological appearance of the mouse hind footpad after a subcutaneous injection with Carr stained with H&E stain at the 5th hr to reveal hemorrhage, edema, and inflammatory cell infiltration in control mice, Carr-treated mice demonstrating hemorrhage with moderately extravascular red blood cells and a large amount of inflammatory leukocyte mainly neutrophils infiltration in the subdermis interstitial tissue of mice, and mice given Indo (10 mg/kg) before Carr. AHT (1.0 g/kg) significantly shows morphological alterations (100x) (a) and the numbers of neutrophils in each scope (400x) compared to subcutaneous injection of Carr only (b).###P<0.001 as compared with the control group. ***P<0.001 compared with the Carr group. Scale bar = 100 μm (one-way ANOVA followed by Scheffe’s multiple range test).
(a)
(b)


## 4. Discussion

In the present study, we demonstrated the anti-inflammatory activities of AHTin vitro and in vivo in experimental systems, using LPS-stimulated RAW264.7 macrophages and Carr-induced paw edema model. Dual inhibitory activities against iNOS and COX-2 as shown in in vitro assays appeared to confer on AHT a potent in vivo efficacy in mouse, Carr-induced, paw edema, comparable with a potent COX inhibitor, Indo, suggesting its potential therapeutic usage as a novel topical anti-inflammatory source of health food.Lipopolysaccharide (LPS) is a prototypical endotoxin derived from Gram-negative bacterial membrane, and it is the initial stimulus leading to induce septic shock syndrome [17]. LPS can directly activate macrophages, endothelial cells, and the complement-triggering production of inflammatory mediators, such as, NO, TNF-α, interleukins (ILs), and leukotrienes [18]. The pharmacological reduction of LPS-inducible inflammatory mediators is regarded as one of the essential conditions to alleviate a variety of disorders caused by activation of macrophages. Thus, RAW264.7 macrophages provide us with a good model for anti-inflammatory drug screening and for subsequently evaluating the inhibitors of the signal pathways that lead to the induction of pro-inflammatory enzymes and to the production of pro-inflammatory cytokines [19].A large amount of NO is produced fromL-arginine by iNOS, thereby causing detrimental effects. Although physiological NO production has beneficial microbicidal, antiparasite, and antitumour effects, excessive NO produced by iNOS is a mediator of the inflammatory diseases and causes cell injury by generating reactive radicals, such as, peroxynitrite [20]. In vitro models, such as, macrophage cells or other cell lines are useful materials with a steady high-level production of NO. The mechanisms by which AHT inhibits macrophage functions have not been elucidated. Results in vitro showed that AHT suppressed LPS-induced production of NO, the expression of inflammatory protein products, such as, iNOS and COX-2. Examination of the cytotoxicity of AHT in RAW264.7 macrophages using MTT assay have indicated that AHT even at 1,000 μg/mL did not affect the viability of RAW264.7 cells. Therefore, the inhibition of LPS-induced nitrite production by AHT was not the result of a possible cytotoxic effect on these cells.LPS elicits strong immune responses, including the production of NO, PGE2, and TNF-α in macrophages. Excess amounts of NO and PGE2 play a critical role in the chronic inflammatory diseases, such as, inflammatory hepatic dysfunction and inflammatory pulmonary disease [21]. Recently, in vitro and in vivo have indicated an existing cross talk between the release of NO and prostaglandins (PGs) in the modulation of molecular mechanisms [22]. While the production of NO and PGE2 was blocked by the NOS inhibitors in mouse macrophages RAW264.7 cells, these inhibitory effects were reversed by coincubation with the precursor of NO synthesis, L-Arginine [23]. In the studies of the mechanism on the inflammation, NO plays an important role in vitro and in vivo. Our present results confirm that LPS-stimulated RAW264.7 macrophages and 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. The level of NO was decreased significantly by treatment with AHT. We suggest the anti-inflammatory mechanism of AHT may be through the L-arginine-NO pathway because AHT significantly inhibits the NO production.TNF-α is another major mediator in inflammatory responses, inducing innate immune responses by activating T cells and macrophages and stimulating secretion of other inflammatory cytokines [15]. In LPS-inducible tissue injury and shock, TNF-α is therefore thought to be a principal mediator. TNF-α induces a number of physiological effects including septic shock, inflammation, and cytotoxicity [21]. 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 [6]. In this study, we found that AHT decreased the TNF-α level in serum after Carr injection.In inflammation, PGE2 is a particular interest because it is involved in all processes leading to the classic signs of inflammation: redness, swelling, and pain [24]. PGE2 is considered as one of the strongest inflammatory mediators in inflammatory response. It was transformed from arachidonic acid via the COX-2 catalytic reaction. Nonsteroidal anti-inflammatory drugs (NSAIDs), which were used widely in current clinical, play their antipyretic, anti-inflammatory and analgesic effects through the inhibition of COX activity and the reduction of inflammatory mediator production, such as, PGE2 [25]. Our study showed that AHT could inhibit PGE2 production and reduce the COX-2 expression significantly in LPS-stimulated Raw264.7 cells and Carr-induced mice paw edema suggested that the anti-inflammatory effect of AHT might be attributed to its inhibitive effect on PGE2 production through blocking COX-2 protein expression.HO-1 catabolizes the rate limiting step in the degradation of cellular heme and produces three products, including CO, biliverdin (which is rapidly converted to bilirubin) and free irons (Fe2+) [26]. HO-1 is currently believed to serve as a protective gene and has anti-inflammatory, anti-apoptotic, and antiproliferative activities. Although the exact mechanisms involved in the anti-inflammatory effects of HO-1 have not been evaluated, HO-1 byproducts have been investigated as possible factors that inhibit macrophage-mediated inflammation [27]. Induction of HO-1 is modulated by various natural and synthetic compounds in tissue culture models. Quercetin, naringin, and hesperidin inhibit LPS-stimulated NO production in accordance with the induction of HO-1 in mouse macrophage-cell lines [28].The Carr-induced mice paw edema is a suitable test for evaluating anti-inflammatory drugs and has frequently been used to assess the antiedematous effect of natural products [29]. Edema formation due to Carr in the mice paw is the biphasic event. The initial phase is attributed to the release of histamine and serotonin. The second phase of edema is due to the release of prostaglandins, protease, and lysosome. The second phase is sensitive to most clinically effective anti-inflammatory drugs. The degree of swelling of the Carr-injected paws was maximal 3th h after injection [2]. AHT and Indo significantly inhibited the development of edema at the 4th and the 5th h after treatment (P<0.001). They both showed anti-inflammatory effects in Carr-induced mice edema paw.The Carr-induced inflammatory response has been linked to neutrophils infiltration and the production of neutrophils-derived free radicals, such as, hydrogen peroxide as well as the release of other neutrophils-derived mediators [30]. Inflammatory effect induced by Carr is associated with free radical. Free radical, prostaglandin, and NO will be released when administrating with Carr for 1–5 h. Reactive oxygen species (ROS) play an important role in modulating the extent of inflammatory response and consequent tissue and cell injury [31]. MDA is a metabolic product of lipid peroxidation, the level of which is raised in oxidative stress. MDA production is due to free radical attack plasma membrane. Increasing evidence regarding free radical-generating agents andinflammatoryprocesses suggests thataccumulationof ROS can cause tissue injury [3]. Thus, inflammatory effect would result in the accumulation of MDA. In this study, there is significantly increased in CAT, SOD, and GPx activities with AHT treatment. Furthermore, there are significantly decreases in MDA level with AHT treatment. We assume the suppression of MDA production is probably due to the increases of CAT, SOD, and GPx activities in the paw edema.Phytochemical and HPLC assay of the AHT led to a conclusion that this active extract part contains plenty of phenolic compounds, especially gallic acid,p-Coumaric acid, and ferulic acid. Phenolic compounds are substances that possess an aromatic ring bearing hydroxyl substituents, including their functional derivatives, such as, esters and methoxy compounds. They exhibit several pharmacological functions such as antioxidant, anti-inflammatory, and anti-cancer [32]. Gallic acidinhibits histamine release and proinflammatory cytokine production in mast cells [33]. p-Coumaric acid and ferulic acid inhibited macrophageinflammatoryprotein-2 production in LPS-stimulatedRAW264.7cells [34]. Then caffeic acid and caffeic acid derivatives exert in vitro and in vivo anti-inflammatory actions, being their actions mediated, at least by the scavenging of NO and their ability to modulate iNOS expression and probably that of other inflammatory mediators [35]. Syringic acid significantly suppressed the cytokineTNF-α, interferon-gamma, and interleukin-6 on concanavalin a-induced liver injury [36]. These phenolic components in the AHT could also play important roles in anti-inflammatory activity. These results suggest that the anti-inflammatory activities of AHT are related to their phenolic compounds.In conclusion, these results suggested that AHT possessed anti-inflammatory effects. The anti-inflammatory mechanism of AHT may be related to iNOS, COX-2, HO-1, and it is associated with the increase in the activities of antioxidant enzymes (CAT, SOD, and GPx). AHT may be used as a pharmacological agent in the prevention or the treatment of disease in which free radical formation is in a pathogenic factor.



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*Source: 132859-2012-06-21.xml*