Running title: Pharmcological Activities of Centipeda minima
Antioxidant and Anti-inflammatory Activities of Aqueous Extract of
Centipeda minima
Shyh-Shyun Huang1, Chuan-Sung Chiu2, Tsung-Hui Lin3, Min-Min Lee4, Chao-Ying Lee1, Shu-Jen Chang1, Wen-Chi Hou5, Guan-Jhong Huang6, *, Jeng-Shyan Deng4, * 1School of Pharmacy, College of Pharmacy, China Medical University, Taichung 404,
Taiwan
2Nursing Department, Hsin Sheng College of Medical Care and Management, Taoyuan 325, Taiwan
3Department of Leisure, Recreation & Holistic Wellness, MingDao University, ChangHua 523, Taiwan
4Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan
5 Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan 6Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources,
College of Pharmacy, China Medical University, Taichung 404, Taiwan
*Corresponding author
Dr. Guan-Jhong Huang, School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan; Tel.: +886-4-2205 3366 ext 5508; Fax: +886 4 2208 3362.
E-mail address: [email protected]
Dr. Jeng-Shyan Deng, Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan; Tel: +3456 ext. 1836. Fax: 886-4-2332-1162; E-mail address: [email protected]
Factors involved in the inflammatory process include physical and chemical stimulants that are released during the immune response and by tissue damage. Reactive oxygen species (ROS) play a significant role in the inflammatory response. During an inflammatory response, the excessive production of ROS can cause major damage to cells, which can lead to DNA damage and mutations. The resulting oxidative stress may lead to aging, inflammation, and other chronic diseases (Wen et al, 2011). Undoubtedly, medicinal plants are relevant in the world as sources of drugs or herbal extracts for various chemotherapeutic purposes. The use of plant derived natural compounds is also as parts of herbal preparations used as alternative sources of medicaments and continues to play major roles in the general wellness of people. Several anti-inflammatory, neuroprotective, and hepatoprotective drugs have been shown to have an antiradical scavenging mechanism as part of their activity (Chang et al., 2011a).
Macrophages are the primary pro-inflammatory cells that provide the first line of defense against these harmful stimuli. In response to extracellular stimuli such as lipopolysaccharide (LPS) stimulation, macrophages mediate the inflammatory response by releasing a variety of pro-inflammatory mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, which exert effects on endothelial and epithelial cells in the local microenvironment (Huang et al., 2011). During inflammation, high levels of ROS were also produced to exert a defense against pathogens. Nitric oxide (NO) production is mainly catalyzed by nitric oxide synthase (NOS) which exists in three isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). eNOS and nNOS are constitutively expressed and play an important role in normal physiological activities. The iNOS-mediated NO production can promote pathological inflammation. Therefore, selective inhibition on iNOS activity has been established as a therapeutic approach for treating inflammation (Deng, et al, 2011).
Centipeda minima (L.) A. Braun & Ascherson is an annual herbaceous plant widely distributed in China, Taiwan, and eastern tropical Asia. The whole plant is traditionally used in Chinese folk medicine for treatment of rhinitis, sinusitis, relieving pain, reducing swelling and treating cancer for a long history (Gan, 1993). Pharmacological studies showed that C. minima possessed anti-tumor (Su et al., 2009), anti-mutagenic (Lee and Lin, 1988), anti-allergic (Liu et al., 2005), anti-protozoal (Yu et al., 1994), anti-bacterial (Liang et al., 2007), and liver protective activities (Qian et al., 2004). Phytochemical studies of its composition have led to the identification, including sesquiterpene lactones, triterpenes, and flavonoids (Wu et al., 1991). In this study, we has showed some physiological effects, but there are no studies focusing on its inhibitory effects on the antioxidant, and the mechanism of anti-inflammatory activities of the aqueous extracts of C. minima (ACM) in cell and animal models. Consequently, the objective of the present study is to determine the therapeutically effects of ACM against antioxidant and anti-inflammatory activities and its active compounds.
2. Materials and methods 2.1. Materials
Lipopolysaccharide (LPS, Escherichia coli O127:B8), 1,1-Diphenyl-2-picrylhydrazyl (DPPH), N-(1-naphthyl) ethylenediamine dihydrochloride, sulfanilamide, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), thiobarbituric acid (TBA), 3-[4,5-dimethyl-thiazol- 2-yl]-2,5-diphenyl tetrazolium bromide (MTT), -Carrageenan (Carr), indomethacin (Indo), 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, China Medical University.
2.2. Preparation of the extracts of plant materials
A 100 g sample of C. minima 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 5.1% (w/w).
2.3. Fingerprint Analysis by HPLC
The chromatographic system consisted of a Qaternary Gradient Pump SFD 2100, a SFD 5200 autosampler, a Merck LiChrospher 100 RP-18e column (5 m, 4.0 I.D.×250 mm) and a S-3210 photodiode-array detector (PDA) (Schambeck SFD GmbH, Bad Honnef, Germany). Peak area was calculated using a Schambeck HPLC-GPC-Software. The samples were analyzed by HPLC on a TSK-GEL ODS-80TM column and detected at 280 nm with acetic acid (2.5%, solvent A) and methanol (solvent B). The gradient program started with 4% solvent B for 0 min, then linearly increased to 12% solvent B for another 7 min. This linear gradient was followed by an isocratic elution until 30 min
and reconditioning steps to return to the initial mobile phase condition. The flow rate was 0.6 mL/min, and the injection volumes of standards and samples were 10 μL. Identification was based on retention times and PDA spectra by comparison with commercial standards.
2.4. Phytochemical content and antioxidant activities of crude extracts in vitro 2.4.1. Determination of total polyphenol content
The total polyphenol contents of crude extracts were determined according to the method of Huang et al (2008). 20 μL of each extract was added to 200 μL distilled water and 40 μL of Folin-Ciocalteu reagent. The mixture was allowed to stand at room temperature for 5 min and then 40 μL of 20 % sodium carbonate was added to the mixture. The resulting blue complex was then measured at 680 nm. Catechin was used as a standard for the calibration curve. The polyphenol content was calibrated using the linear equation based on the calibration curve. The total polyphenol content was expressed as mg catechin equivalence (CE)/g dry weight.
2.4.2. Determination of antioxidant activity by ABTS·+ scavenging ability
The ABTS·+ scavenging ability was determined according to the method of Huang et al., (2008). Aqueous solution of ABTS (7 mM) was oxidized with potassium peroxodisulfate (2.45 mM) for 16 hrs in the dark at room temperature. The ABTS·+ solution was diluted with 95% ethanol to an absorbance of 0.75 ± 0.05 at 734 nm (Beckman UV-Vis spectrophotometer, Model DU640B). An aliquot (20 μL) of each sample was mixed with 180 μL ABTS·+ solution and the absorbance was read at 734 nm after 1 min. Trolox was used as a reference standard.
2.4.3. Determination of antioxidant activity by DPPH radical scavenging ability
The effects of crude extracts and positive controls (glutathione) on DPPH radicals were estimated according to the method of Huang et al., (2008). Aliquot (20 μL) of crude extracts at various concentrations were each mixed with 100 mM Tris-HCl buffer (80 μL, pH 7.4) and then with 100 μL of DPPH in ethanol to a final concentration of 250 μM. The mixture was shaken vigorously and left to stand at room temperature for 20 min in the dark. The absorbance of the reaction solution was measured spectrophotometrically at 517 nm. The percentages of DPPH decolorization of the samples were calculated according to the equation: % decolorization = [1- (ABS sample /ABS control)] ×100. IC50 value was the effective concentration at which DPPH radicals were scavenged by 50% and was obtained by interpolation from linear regression analysis.
2.5. Cell culture
A murine macrophage cell line RAW 264.7 (BCRC No. 60001) was purchased from the Bioresources Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in plastic dishes containing Dulbecco’s Modified Eagle Medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, USA) in a CO2 incubator (5% CO2 in air) at 37°C and subcultured every 3 days at a dilution of 1:5 using 0.05% trypsin–0.02% EDTA in Ca2+-, Mg2+- free phosphate-buffered saline (DPBS).
Cells (5 x 104) were cultured in 96-well plates containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. Then cells were cultured with samples in the presence of 100 ng/mL LPS for 24 hrs. After that, the cells were washed twice with DPBS and incubated with 100 L of 0.5 mg/mL MTT for 2 hrs at 37°C testing for cell viability. The medium was then discarded and 100 L dimethyl sulfoxide (DMSO) was added. After 30-min incubation, absorbance at 570 nm was read by using a microplate reader.
2.5.2. Measurement of Nitric oxide/Nitrite
Nitrite levels in the cultured media and serum, which reflect intracellular NO synthase activity, were determined by Griess reaction (Huang et al., 2007). The cells were incubated with samples in the presence of LPS (100 ng/mL) at 37°C for 24 hrs. Then, cells were dispensed into 96-well plates, and 100 L of each supernatant was mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 5% phosphoric acid) and incubated at room temperature for 10 min. With using sodium nitrite to generate a standard curve, the concentration of nitrite was measured form absorbance at 540 nm.
2.6. Animals
This study was conducted in conformity with the policies and procedure details in the “Guide for the Care and Use of Laboratory Animals” (NIH Publication No. 86–23 1985) and was approved by the ethics committee of the Institutional Animal Care and Use Committee (IACUC) of China Medical University, Taichung, Taiwan. ICR strain male mice (6−8 weeks old) were obtained from BioLASCO Taiwan Co., Ltd., Taipei,
Taiwan. The animals were housed in an environmentally controlled room (temperature 22 ± 1 °C; relative humidity 55 ± 5%; 12 h dark–light cycle). They were given food and water ad libitum.
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. The other five groups include a Carr-treated, a positive control (Carr + Indo) and ACM administered groups (Carr + ACM: 25, 50, and 100 mg/Kg).
2.6.1. Determination of carrageenan (Carr) induced edema
Carr-induced hind paw edema model was used for determination of anti-inflammatory activity (Winter et al., 1962). After a 2-week adaptation period, male ICR mice (18 to 25 g) were randomly assigned to five groups (n = 6) including Carr, positive Indo control and three ACM-treated groups. Carr group received 1% Carr (50 μL). ACM at doses of 25, 50, and 100 mg/Kg were orally administered 2 hrs before the injection with 1% Carr (50 μL) in the plantar side of right hind paws of the mice. And Indo (10 mg/Kg) was intraperitoneally administered 90 min before the injection with 1% Carr (50 μL) in the plantar side of right hind paws of the mice. Paw volume was measured immediately after Carr injection at 1, 2, 3, 4, and 5 h intervals using a plethysmometer (model 7159, Ugo Basile, Varese, Italy). The degree of swelling induced was evaluated by a minus b, where a was the volume of the right hind paw after Carr treatment and b was the volume of the right hind paw before Carr treatment. Indo was used as a positive control.
In the later experiment, the right hind paw tissue was taken at the 5th h. The right hind paw tissue was rinsed in ice-cold normal saline, and immediately placed in cold normal saline four times their volume and homogenized at 4 ºC. Then the homogenate
was centrifuged at 12,000×g for 5 min. The supernatant was obtained and stored at −20 ºC refrigerator for MDA and the antioxidant enzymes (CAT, SOD, and GPx) activities assays.
2.6.2. Determination of tissue lipid peroxidation
MDA was evaluated by the thiobarbituric acid reacting substances (TRARS) method (Deng et al., 2012). 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.
2.6.3. Measurement of tumor necrosis factor (TNF-α) and interleukin-1 (IL-1) in serum
Serum levels of TNF-α and IL-1β were determined using a commercially available ELISA kit (Biosource International, Inc., Camarillo, CA) according to the instructions of the manufacturer. TNF-α and IL-1β were determined from a standard curve. The concentrations were expressed as pg/mL.
2.6.4. Determination of antioxidant enzyme activity in paw tissue
The following biochemical parameters were analyzed to check the protective activity of ACM 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 requiredto inhibit the rate of cytochrome c reduction by 50%. Total CAT
activity estimation was based on the previously reported (Armstrong & Browne, 1994). In brief, the reduction of 10 mM H2O2 in 20 mM of phosphate buffer (pH 7) was monitored by measuring the absorbance at 240 nm. The activity was calculated by using a molar absorption coefficient, and the enzyme activity was defined as nanomoles of dissipating hydrogen peroxide per milligram protein per minute. Total GPx activity in cytosol was determined as previously reported (Flohe & Gunzler, 1984). The enzyme solution was added to a mixture containing hydrogen peroxide and glutathione in 0.1 mM Tris buffer (pH 7.2) and the absorbance at 340 nm was measured. Activity was evaluated from a calibration curve, and the enzyme activity was defined as nanomoles of NADPH oxidized per milligram protein per minute. The protein concentration of the tissue was determined by the Bradford dye-binding assay (Bio-Rad, Hercules, CA).
2.6.5.Protein Lysate Preparation and Western blot Analysis of iNOS and COX-2
Total protein was extracted with a RIPA solution (radioimmuno-precipitation assay buffer) at -20°C overnight. We used BSA (bovine serum albumin) as a protein standard to calculate equal total cellular protein amounts. Protein samples (30g) were resolved by denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using standard methods, and then were transferred to PVDF membranes by electroblotting and blocking with 1% BSA. The membranes were probed with the primary antibodies (iNOS, COX-2, and -actin) at 4°C overnight, washed three times with PBST, and incubated for 1 h at 37 °C with horseradish peroxidase conjugated secondary antibodies. The membranes were washed three times and the immunoreactive proteins were detected by enhanced chemiluminescence (ECL) using hyperfilm and ECL reagent (Amersham International plc., Buckinghamshire, U.K.). The results of Western blot analysis were quantified by measuring the relative intensity compared to
the control using Kodak Molecular Imaging Software and represented in the relative intensities.
2.6.6. Histological examination
For histological examination, biopsies of paws were taken 5th hrs following the interplanetary injection of Carr. The tissue slices were fixed in (1.85% formaldehyde, 1% acetic acid) for 1 week at room temperature, dehydrated by graded ethanol and embedded in Paraffin (Sherwood Medical). Sections (thickness 5 μm) were deparaffinized with xylene and stained with H & E stain. All samples were observed and photographed with BH2 Olympus microscopy. Every 3-5 tissue slices were randomly chosen from Carr, Indo and ACM treated (100 mg/Kg) groups. 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.
2.7. Statistical analysis
Experimental results were presented as the mean ± standard deviation (SD) of three parallel measurements. IC50 values were estimated using a non-linear regression algorithm (SigmaPlot 8.0; SPSS Inc. Chicago, IL). 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
To establish the fingerprint chromatogram for the quality control of ACM. Protocatechuic acid, chlorogenic acid, protocatechualdehyde, vanillic acid, caffeic acid, and ferulic acid were used as markers. An optimized HPLC-PAD technique was employed. HPLC chromatograms showed six marker components present in ACM. As shown in Fig. 1, these phenolic components have been identified as protocatechuic acid, chlorogenic acid, protocatechualdehyde, vanillic acid, caffeic acid, and ferulic acid 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 five constituents and their correlation coefficients (r) were determined as follows: protocatechuic acid, y = 0.622x + 4.301 (r2 = 0.999); chlorogenic acid, y = 0.579x + 3.893 (r2 = 0.999); protocatechualdehyde, y = 0.145x -0.614 (r2 = 0.999); vanillic acid, y = 0.232x+1.786 (r2 = 0.992); caffeic acid, y = 1.643x + 11.86 (r2 = 0.999), ferulic acid, y = 0.286x -0.492 (r2 = 0.999). The relative amounts of the six phenolic compounds found in ACM were in the order of protocatechualdehyde (2.66 mg/g extract) > vanillic acid (2.56 mg/g extract) > protocatechuic acid (1.12 mg/g extract) > chlorogenic acid (0.39 mg/g extract) > ferulic acid (0.21 mg/g extract) > and caffeic acid (0.12 mg/g extract), respectively.
3.2. Trolox Equivalent Antioxidant Capacity (TEAC)
TEAC assay is often used to evaluate the total antioxidant power of single compounds and complex mixtures of various plants (Huang et al., 2010). Table 1 shows TEAC values of ACM. TEAC value of ACM extract was 4.85 ± 0.03 mol TE/g dw. As shown in Table 1, reference compounds protocatechuic acid, chlorogenic acid, protocatechualdehyde, vanillic acid, caffeic acid, and ferulic acid in the ACM showed
TEAC value of 4.32 ± 0.04, 7.48 ± 0.03, 8.23 ± 0.06, 2.43 ± 0.07, 8.67 ± 0.04, and 6.85 ± 0.08 mol TE/g dw, respectively.
3.2.1. Scavenging Activity against 1, 1-Diphenyl-2-Picrylhydrazyl Radical
The organic radical DPPH is widely used in modeling systems to investigate the scavenging activities of several natural compounds, such as phenolic acids, as well as crude extract of plants (Huang et al., 2008). ACM exhibited the strongest antioxidant activities in scavenging DPPH radicals, with IC50 values of 136.39 ± 1.39 mg/mL respectively (Table 1). As shown in Table 1, reference compounds protocatechuic acid, chlorogenic acid, protocatechualdehyde, vanillic acid, caffeic acid, and ferulic acid in the ACM showed DPPH radical scavenging with an IC50 value of 7.85 ± 0.32, 7.89 ± 0.06, 6.35± 0.12, 247.64 ± 1.35, 5.75 ± 0.04, and 21.56 ± 0.13 g/mL, respectively.
3.2.2. Determination of Total Phenolic Content in the Plant Extract
Total phenolic content was expressed as mg of catechin equivalent per gram of dry weight. The results showed that ACM had the highest phenolic contents of 137.67 ± 2.54 mg CE/g, respectively (Table 1).
3.3. Effect of the ACM extract on LPS-induced NO Production in Macrophages
The effect of the ACM extract on RAW264.7 cell viability was determined by a MTT assay. Cells cultured with the ACM extract at the concentrations (0, 12.5, 25, 50, and 100 g/mL) used in the presence of 100 ng/mL LPS for 24 h did not change cell viability, significantly (Fig. 2A). In the present study, effects of the ACM extract, its fractions and reference compounds on LPS-induced NO production in RAW 264.7
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 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 the ACM extract and fractions together with LPS for 24 h, the ACM extract inhibited nitrite production significantly (Fig. 2B). When RAW264.7 macrophages were treated with different concentrations of ACM (0, 12.5, 25, 50, and 100 g/mL) together with LPS (100 ng/mL) for 24 h, a significant concentration-dependent inhibition of nitrite production was detected. The NO inhibitory activity of ACM induced by LPS in RAW264.7 macrophages with an IC50 value of 87.53 ± 1.35 g/mL. The reference compounds of protocatechualdehyde and caffeic acid in the ACM also showed the NO inhibitory activity induced by LPS in RAW264.7 macrophages with an IC50 value of 21.45 ± 0.13 and 28.18 ± 0.16 g/mL, respectively. Protocatechuic acid, chlorogenic acid, vanillic acid, and ferulic acid had weak the NO inhibitory activity induced by LPS in RAW264.7 macrophages, respectively. Whatever, We evaluated the reference compounds in the ACM that protocatechualdehyde and caffeic acid were studies on the pharmacological activities by free radical scavenging and LPS-induced NO production in RAW264.7 macrophages.
3.3.1. Effects of ACM on TNF-α and IL-1 levels
When RAW264.7 macrophages were treated with different concentrations of ACM (0, 12.5, 25, 50, and 100 g/mL) with LPS (100 ng/mL) for 24 hours, a significant concentration-dependent inhibition of TNF-α and IL-1 levels were detected. There was either a significant decrease in the nitrite production of group treated with 25 g/mL
ACM (p < 0.05), or highly significant decrease of groups treated respectively with 50 and 100 g/mL of ACM when compared with the LPS-alone group (p < 0.01 or p < 0.001). The IC50 value for inhibition of TNF-α and IL-1 levels of ACM were about 78.64 ± 2.35 and 82.35 ± 2.71 g/mL (Fig. 2C and 2D).
3.3.2. Inhibition of LPS-induced iNOS and COX-2 Protein by ACM
The results showed that incubation with ACM in the presence of LPS for 24 h inhibited iNOS and COX-2 protein expressions in mouse macrophage RAW264.7 cells in a dose-dependent manner (Fig. 3A). The intensity of protein bands were analyzed and showed an average of 80.2% and 71.2% down-regulation of iNOS and COX-2 proteins, respectively after the treatment with ACM fraction at 100 g/mL compared with the LPS-alone (Fig. 3B).
