Journal of Ethnopharmacology 153 (2014) 793–800

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Research Paper

Antioxidant effects of Genkwa flos flavonoids on Freund's adjuvant-induced rheumatoid arthritis in rats Chun-Feng Zhang a,b,n, Su-Li Zhang a, Xin He c, Xiao-Lin Yang d, Hai-Tao Wu a, Bao-Qin Lin e, Cui-Ping Jiang a, Jun Wang a, Chun-Hao Yu b, Zhong-Lin Yang a, Chong-Zhi Wang b, Ping Li a, Chun-Su Yuan b a

State Key Laboratory of Natural Medicines, China Pharmaceutical University, No. 24 Tongjia Lane, Nanjing 210009, China Tang Center of Herbal Medicine and Department of Anesthesia & Critical Care, University of Chicago, Chicago 60637, USA c School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210000, China d Guangdong Provincial Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, China e Department of Pharmacology, School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, China b

art ic l e i nf o

a b s t r a c t

Article history: Received 9 October 2013 Received in revised form 17 March 2014 Accepted 19 March 2014 Available online 28 March 2014

Ethnopharmacological relevance: Genkwa flos (Daphne genkwa Sieb. et Zucc.), a Chinese herbal medicine, has been traditionally used for over two thousand years in China for inflammation related symptoms, including joint pain. To evaluate the antioxidative effects of flavonoid aglycones (FA) isolated from Genkwa flos on adjuvant arthritis in rats and to identify the relationship between antioxidant potential and whole blood viscosity (WBV). Materials and methods: FA compounds were identified using LC–MS and the content was assayed by HPLC. Arthritis was induced by an intradermal injection of Freund's complete adjuvant in the footpad. The effects of FA on paw volumes, secondary arthritis scores, histopathology of joints, and body and organ weights were measured. The antioxidant effects of FA and WBV were determined. Results: LC–MS analysis showed that the FA contained four major compounds: luteolin, apigenin, hydroxygenkwanin and genkwanin. FA significantly decreased paw edema, arthritis scores, and weight loss. These observations were consistent with the reduction of oxidative stress and the improvement of the WBV. Conclusion: FA significantly decreased arthritis in a rat model through antioxidant and hemorheological modulatory mechanisms. The Genkwa flos flavonoids may have clinical potential for the treatment of rheumatoid arthritis. & 2014 Elsevier Ireland Ltd. All rights reserved.

Chemical compounds studied in this article: Luteolin (PubChem CID: 5280445) Apigenin (PubChem CID: 5280443) Hydroxygenkwanin (PubChem CID: 5320946) Genkwanin (PubChem CID: 5281617) Keywords: Genkwa flos Flavonoid aglycone Rheumatoid arthritis Antioxidant Hemorheology

1. Introduction

Abbreviations: FA, flavonoid aglycones from Genkwa flos; WBV, whole blood viscosity; RA, rheumatoid arthritis; TCM, traditional Chinese medicine; Dex, dexamethasone; FCA, Freund's complete adjuvant; AIA, adjuvant-induced arthritis; AI, arthritis index; PS, poly-arthritis score; SACS, secondary arthritis composite scores; ANOVA, analyzed by one-way analysis of variance; CTR, control group; MOD, model group; NO, nitric oxide; MDA, malondialdehyde; T-AOC, total antioxidant capacity; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; TNF-α, tumor necrosis factor-α; IL-6, interleukin- 6; iNOS, inducible isoform of nitric oxide synthase; COX-2, cyclooxygenase-2; NF-κB, nuclear factor-kappa B; IFN-γ, interfern-γ; APCs, antigen-presenting cells n Corresponding author at: State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjia Lane, Nanjing, Jiangsu 210009, China. Tel.: þ 86 25 86185129. E-mail addresses: [email protected], [email protected] (C.-F. Zhang). http://dx.doi.org/10.1016/j.jep.2014.03.046 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

Rheumatoid arthritis (RA), an autoimmune disease, has characteristic pathological changes of persistent synovitis, vascular proliferation, infiltration of inflammatory cells, as well as damage of cartilage and bone (Lee et al., 2009; Pan et al., 2009; Xu et al., 2013a). Large amounts of oxygen free radicals produced by macrophages have a close relationship with joint injury (Hadjigogos, 2003; Lund-oleson, 1970). In traditional Chinese medicine (TCM) theory, RA-related symptoms belong to the category of Bi Zheng, which can be manifested as arthralgia and dyskinesia of the joints and limbs due to an attack of the meridians of the limbs by wind, dampness, and heat or cold pathogens (Lam et al., 2008). The marked symptoms of this hemorheological disorder include the decrease in blood flow velocity, disturbance of the normal blood flow, also expressed in TCM as Bi Zheng (Li et al., 2009). Recent studies have

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shown that RA patients have a significant hemorheological disorder-blood hypercoagulable state (Maria et al., 2011) and barrier of microcirculation, which is consistent with the Chinese medicine theory “blood stasis is throughout the RA disease process.” The treatment of activating blood circulation to dissipate blood stasis is an important therapeutic principle in clinically improving the symptoms of RA (Balabanova et al., 1990; Li et al., 2009). Genkwa flos, the flower buds of Daphne genkwa Sieb. et Zucc., belongs to the Thymelaeaceae family. It was initially described in “Shennong's Herbal Classic” (Shen Nong Ben Cao Jing) published over two thousand years ago, widely considered as the oldest book on oriental herbal medicine and the foundation of TCM (Li et al., 2007). Based on TCM, the properties of Genkwa flos are a pungent and bitter flavor, warm nature, poisonous, and activity in the lung, kidney and large intestine meridians (Zhang et al., 1988). Genkwa flos can remove fluid retention, eliminate phlegm, and improve skin and joint disorders by draining and expelling out congested fluids from the body. As a traditional Chinese herb, it has been used to alleviate rheumatalgia (Chang et al., 2012). Modern researches have shown that Genkwa flos, containing mainly flavonoids, possesses anti-inflammatory (Lee et al., 2009; Zhang et al., 2010), analgesic (Wang et al., 2005) and immunoregulatory activities (Shi, Zheng 2004; Zheng et al., 2004; Gao et al., 2006). In this study, we evaluated the antioxidant effects of Genkwa flos flavonoids on adjuvant-induced arthritis in rats, and investigated the relationship between antioxidant potential and hemorheology modulatory mechanisms.

2. Materials and methods

performance liquid chromatography–mass spectrometry (HPLC–MS) and the content of the compounds was assayed with HPLC. Analyses were performed on an Agilent Series 1100 liquid chromatography (Agilent Technologies, Palo Alto, CA, USA), consisting of a dual pump, an autosampler, a DAD coupled with an ELSD (Alltech Associates, Deerfield, USA), HP ChemStation software (Agilent Technologies, Palo Alto, CA, USA), and a Zorbax ODS C18 column (250 mm  5 μm). The column temperature was kept constant at 25 1C, the injection volume was 20 μL, and the mobile phase flow rate was 1 mL/min. The mobile phases consisted of 65% methanol (A) and 35% water (B). An Agilent 1100 Series LC/MSDSL G1946D quadruple mass spectrometer equipped with an ESI source and running Agilent Chem-Station software was used for HPLC–MS analysis. ESI–MS conditions were as follows: positive ion mode; drying gas (N2), 10 mL/min; nebulizer (N2), 35 psi (241 kPa); drying gas temperature, 320 1C; capillary voltage, 4000 V; fragmentation voltage, 100 V; mass range (m/z), 100–1500. 2.4. Animals The experiments were performed in accordance with the Animal Ethics Committee of China Pharmaceutical University. Male Sprague Dawley (SD) rats (150–180 g) were purchased from Zhejiang Province Experimental Animal Center (Zhejiang, China). All rats had free access to a standard diet and drinking water, and were housed in an animal room maintained at 26.0 70.5 1C and with a 12:12 h cyclic lighting schedule. They were housed in cages in which the floor was covered with sawdust to minimize the possibility of painful contact with a hard surface. Animals were divided into four groups: Control, Model, Dex and FA, and there were more than eight rats per group.

2.1. Plant materials 2.5. Adjuvant-induced arthritis (AIA) The raw materials of the flower bud of Daphne genkwa Sieb. et Zucc. were collected in October 2010 from Anhui Province, China. The materials were identified by Professor Ping Li, School of Traditional Chinese Medicine of China Pharmaceutical University. A voucher specimen (No. 23586) was deposited in the herbarium of China Pharmaceutical University. All the materials were dried at room temperature to a constant weight. 2.2. Chemicals and reagents All chemicals were of analytical grade. Petroleum ether and methanol were purchased from Nanjing Hanbang Chemical Reagent Company (Nanjing, China). Reference luteolin, apigenin, hydroxygenkwanin and genkwanin were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity of each standard compound was determined to be more than 98% by HPLC. Mycobacterium butyricum was purchased from Difco laboratories (Detroit, MI, USA), and dexamethasone (Dex) was obtained from Zhejiang Xianju Pharmaceutical Co. Ltd. (Xianju, China). SOD, MDA, NO, GSH-Px and T-AOC kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 2.3. Isolation, purification and identification of FA The powdered flower buds of Daphne genkwa Sieb. et Zucc. (40 meshes) were refluxed with ethanol three times. Following the evaporation of the ethanol in a rotary evaporator, the residue was partitioned with ligroin and aqua. The remaining residue was purified with column chromatography over versamide (100 meshes) with a gradient of ethanol and water as eluent to obtain the FA. The main compounds of FA were identified by high-

Rats were injected intradermally into the plantar skin of the right hind paw with a single dose of 100 μl Freund's complete adjuvant (FCA) containing 10 mg lyophilized M. butyricum in 1 ml liquid paraffin (Lam et al., 2008; Lee et al., 2009). In the control group, the same volume of liquid paraffin alone was given. AIA induction day was designed as day 0. FA (8.3 mg/ml) and Dex (0.1 mg/ml) were suspended in 0.5% CMC–Na solution. FA (50 mg/kg) and Dex (0.6 mg/kg) were administered orally from day 20 when secondary case arthritis was obvious and was continued for the next 15 days. Rats in the control and model groups were orally administered 0.5% CMC–Na. 2.5.1. Assessment of paw volumes and arthritic score The hind-paw volumes were measured via the water displacement method with a plethysmometer. The measurement of the Arthritis Index (AI): An evaluation of joint inflammation was performed by a blinded independent observer with no knowledge of the treatment protocol. The severity of arthritis in each paw was quantified every 5 days from the 20th day by a clinical score measurement of 0–4 as follows: 0 – no macroscopic signs of arthritis (swelling or erythema), 1 – swelling or erythema (wrist or ankle joints), 2 – moderate swelling, 3 – severe swelling, 4 – severe swelling with no weight bearing ability. The maximum score for each rat was 12. Assay of Poly-arthritis Score (PS): Symptoms of ear erythema and tail rheumatoid nodule were recorded as secondary lesions in AIA rats because of the delayed hypersensitivity. The PS was assessed from the 20th day of the experiment and as follows: 0 – no macroscopic signs of ear erythema or tail rheumatoid nodule, 1 – ear erythema or tail rheumatoid nodule, 2 – ear erythema and tail rheumatoid nodule.

C.-F. Zhang et al. / Journal of Ethnopharmacology 153 (2014) 793–800

Secondary Arthritis Composite Scores (SACS) were determined on days 20, 25, 30 and 35, and calculated as the formula of AI plus PS. 2.5.2. Histological assessment of joints Knee joints were isolated after the animals were sacrificed. Samples were then fixed in 10% buffered formalin for 24 h, and then decalcified in 10% formic acid solution. After decalcification, the joints were dehydrated, processed and embedded in paraffin wax (melting point was 56 72 1C). Five micrometer thick sections from each block were stained with haematoxylin and eosin, and then evaluated under a compound microscope. 2.5.3. Measurement of body weight and immune organ weight The body weight of each animal was measured every other day throughout the experiment. Rats were anesthetized with ether 1 h after the last administration on day 36. Blood was collected into heparinized (20 U/ml) and non-heparinized tubes from the femoral artery. The liver, spleen, thymus glands and adrenal glands were removed after the animals were sacrificed, and then weighed immediately after rinsing with ice-cold saline solution. 2.5.4. Toxicity/adverse effects assessment Animals were closely monitored for unusual behavioral changes and symptoms, such as obvious temperature change, diarrhea, weight loss, fur discoloration, lethargy, irritation and convulsion, during the 15 days of administration. After the observation period, all animals were euthanatized and a comprehensive necropsy was performed. All observations were made by investigators who were blinded to the group assignment. 2.6. Measurement of whole blood viscosity (WBV) Eight hundred microliters of heparinized blood was used to measure WBV with an automatic viscometer (Model EBY-N6A, Zhongxi Co., Beijing, China) at shear rates of 10 s  1 and 80 s  1. 2.7. Biochemical assays Non-heparinized blood was centrifuged at 4000 r/min for 10 min, and the supernatant was collected. The levels of T-AOC, MDA and NO, and the activities of SOD and GSH-Px in the serum were measured according to the manufacturer's instructions. 2.8. Statistical analysis Data were expressed as mean 7SE. Values were analyzed by one-way analysis of variance (ANOVA) using SPSS version 18.0 software. A P value of less than 0.05 was considered significant.

3. Results 3.1. Analysis of FA As shown in Figs. 1A and 2A, peak 1 was identified as luteolin by comparison with the reference compound. Peak 1 mainly gives the [M þ ] (m/z: 284.9). It also can be seen in Fig. 1B and Fig. 2A that there is the [M þ ] (m/z: 268.9) in peak 2, assigned to apigenin. However, in Fig.1C and Fig. 2A, peak 3 gives the [M þ ] (m/z: 298.9) and the fragment ion [M-15 þ ] (m/z: 283.8), which belong to hydroxygenkwanin. Similarly, in Fig. 1D and Fig. 2A, peak 4, including the [M þ ] (m/z: 282.9) and the fragment of ion [M-15 þ ] (m/z: 267.8), was identified as genkwanin.

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Therefore, luteolin, apigenin, hydroxygenkwanin and genkwanin were the four main compounds in FA, and the weight proportions were 6.5%, 19.9%, 32.5% and 31.9%, respectively. The total content of the four compounds was about 90.8% (Fig. 2B). 3.2. Therapeutic effects of FA on AIA rats In the model group, paw edema increased remarkably following the onset of the secondary phase arthritis and peaked around day 19. Although FA had no highly significant effects on right paw volume (Fig. 3B) (P o0.05 vs. model group), it significantly lowered the left paw volumes of the rats from day 29 to 35 (P o0.01 vs. model group). On day 35, the left paw swelling volumes of rats in the FA group were 68.2% of those of the model group (Fig. 3A). The arthritis score of the model group increased gradually and then peaked around 20 days after the CFA injection. FA gradually inhibited the arthritis of rats. The FA group had 4.5 SACS less than the model group on day 35 (P o0.001, Fig. 3C). The body weight of rats in the model group was significantly less than that of the control group in the autoimmune course (Fig. 3D). The body weight of FA-treated rats was higher than that of the model group from day 23 to 35. The body weight of the FA group was 38.5 g higher than that of the model group on day 35. However, the rats of Dex group weighed 36.0 g less than the rats of the model group. The histological architecture of joints in the model group rats was markedly abnormal with immune cell infiltration, tissue proliferation, increased number of vessels and erosion of cartilage and bone. The joints of the Dex and FA groups were significantly different from those of the model group and moved toward to those of the control group (Fig. 4). The liver, spleen, thymus and adrenal gland are main immune system organs. Their relative organ weights are important indicators reflecting immunological function (Fig. 5). There is a significant difference in weight of the organs between the model group and control group (P o 0.001 vs. control group). FA restored the liver and thymus weight to a normal value. FA could also reverse the spleen weight (P o 0.01 vs. model group), but both Dex and FA had no effect on adrenal weight (P 4 0.05 vs. control group). No significant behavioral changes or obvious symptoms except signs associated with the FCA injection were observed in either the model or treatment groups during the 15 days of daily administration. Moreover, stomach ulcers or bleeding foci were not found in any animals in the necropsy study. 3.3. Antioxidant effects of FA on AIA rats Fig. 6A showed that the levels of nitric oxide (NO) and malondialdehyde (MDA) were both increased significantly in arthritic animals, whereas the levels of all the endogenous antioxidants [total antioxidant capacity (T-AOC), superoxide dismutase (Cu/Zn-SOD) and glutathione peroxidase (GSH-Px)] were found to be decreased in arthritic animals. FA significantly reversed the above changes. FA significantly decreased the NO level to 70.5% of that of model group. Both FA and Dex could restore the MDA level to the normal range. FA significantly reduced the level of MDA to 80.0% of that of the model group. FA (50.0 mg/kg) restored the T-AOC level to normal, and even 5.10% higher than the level of the control group. Although FA had no regulative effect on the activities of total SOD and Mn-SOD, it could significantly increase the activity of Cu/Zn-SOD to 141.5% of the model group, and 20.05% higher than the control group. Treatment with FA increased the activities of GSH-Px to 111.7% of that of the model

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Fig. 1. LC–MS of flavonoid aglycones (FA). (A) luteolin; (B) apigenin; (C) hydroxygenkwanin; and (D) genkwanin.

group, whereas Dex had no obvious effect on GSH-Px activity (Fig. 6B). 3.4. Effects of FA on whole blood viscosity (WBV) and its correlation to antioxidant levels As seen in Fig. 7, WBV of model group was significantly increased (Po0.001 vs. control) at both a low shear and high shear rate. FA, at the dose of 50.0 mg/kg, significantly reversed the viscosity compared

to the model group at a low shear rate (Po0.01). At a low shear rate (10 s  1) and high shear rate (80 s  1), the WBV of the FA group was 94.86% (Po0.01) and 92.32% (Po0.01) of the model group, respectively (Fig. 7A). The level of NO had a strong direct correlation with WBV at high shear rate, 0.9419. The activity of GSH-Px had a negative correlation with the WBV, from the low shear rate to the high shear rate the correlation coefficients were  0.5048 and  0.8175, with a gradual increase in linear correlation (Fig. 7B).

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Fig. 2. Mass spectrum and HPLC chromatogram of flavonoid aglycones (FA). (A) LC–MS (A1) and LC–UV (A2) chromatograms of FA. Compounds were identified through the comparison of retention times and molecular ions with corresponding standards. (B) The percentage of luteolin, apigenin, genkwanin and hydroxygenkwanin in the FA. Their percentages were 6.5%, 19.9%, 31.9% and 32.5%, respectively.

Fig. 3. Pharmacological effects of FA on adjuvant-induced arthritis rats. (A) Left paw volume in rats, (B) right paw volume in rats, (C) comparison of secondary arthritis composite score, and (D) body weight changes. nPo 0.05, nnP o 0.01, nnnPo 0.001 vs. model group; &P o 0.05, &&P o0.01, &&&Po 0.001 vs. control group.

4. Discussion RA is associated with systemic inflammatory disorders involving multiple joints. It is an autoimmune disorder of unknown etiology that is characterized by progressive joint destruction, deformity, disability and premature death in most patients (Lee et al., 2009; Chen et al., 2013). It is pathologically characterized by synovial hyperplasia, inflammatory cell infiltration and angiogenesis (Pan et al., 2009; Xu et al., 2013b). CFA-induced secondary inflammation mimics sub-acute RA characterized by excessive immunologic activity in the synovium (Fan et al., 2005). FA, at the dosage of 50.0 mg/kg, significantly decreased inoculated paw volume (Fig. 3A) and SACS (Fig. 3C), inhibited the decrease of body weight (Fig. 3D) and modulated the relative immune organ weight (Fig. 4). These changes indicated that FA had antiinflammatory, anti-rheumatic and immunoregulatory activities in CFA-stimulated chronic arthritis in rats. In particular, FA showed a better therapeutic effect on the second signal of left hind paw edema from an immunological reaction compared to

those of the first signal of the right hind paw edema after injection of CFA in rats, which demonstrated that the therapeutic effect of FA on AIA rats mainly is through immunoregulation. Dex significantly inhibited the arthritis of the two-phase response of the AIA rat. However, the Dex-treated rats lost body weight acutely, which may be caused by the immunosuppression effect of Dex. Histological assessment of a joint section from rat ankle was also conducted. It was demonstrated that FA could improve synovial hyperplasia, reduce the infiltration of inflammatory cells in the synovium, and diminish erosive damage in the cartilage and bone as well (Fig. 5). These joint histological results were consistent with the observations that FA is capable of reducing clinical symptoms of AIA rat. Luteolin, a plant flavonoid, has potent antiinflammatory properties both in vitro and in vivo. Luteolin suppresses the gene expression of LPS-induced TNF-α, IL-6, iNOS and COX-2 at a transcriptional level, and reduces the DNA binding activity of nuclear factor-kappa B (NF-κB) in LPS-activated macrophages as well (Chen et al., 2007;

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Fig. 4. Representative histological sections of rat ankle joints from different experimental groups. (A) Control; (B) Model; (C) DX; and (D) FA.

Fig. 5. Changes in immune organ weight in adjuvant-induced arthritis rats. nPo 0.05, control group.

Xagorari et al., 2001). Moreover, the flavones luteolin and apigenin were found to be strong inhibitors of both murine and human T-cell responses and significantly reduced antigen-specific IFN-γ production other than T-cell proliferation (Verbeek et al., 2004). Since apigenin inhibited the autoantigen-presenting and stimulatory

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functions of antigen-presenting cells (APCs) that are necessary for the activation and expansion of autoreactive Th1 and Th17 cells and B cells in lupus, it could be valuable for suppressing inflammation in lupus, other Th17-mediated diseases like RA, Crohn's disease, and psoriasis, as well as in inflammation-based tumors overexpressing

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Fig. 6. Antioxidant effects of FA in adjuvant-induced arthritis rats. (A) Levels of NO and MDA, and (B) levels of T-AOC, SOD and GSH-Px. nPo 0.05, nnP o 0.01, nnnPo 0.001 vs. model group; &Po 0.05, &&Po 0.01, &&&P o 0.001 vs. control group.

Fig. 7. Effects of FA on WBV and the correlation with antioxidant levels in adjuvant-induced arthritis rats. (A) Changes in whole blood viscosity (WBV) in relation to shear rate and (B) correlation coefficient of the shear rate and antioxidant. nPo 0.05, nnP o 0.01, nnnPo 0.001 vs. model group, &Po 0.05, &&Po 0.01, &&&P o 0.001 vs. control group.

COX-2 (Kang et al., 2009). In addition, genkwanin could significantly inhibit the cotton pellet-induced rat granuloma (Po0.05) (Pelzer et al., 1998). These previous results showed that the compounds in FA, luteolin, apigenin and genkwanin, had anti-inflammatory and immunoregulatory activity. Our results also showed that FA had an anti-RA effect in rats. So luteolin, apigenin and genkwanin might be the effective compounds of FA on RA. One approach to the treatment of RA is to employ antioxidants. Free radicals are chemical species possessing an unpaired electron. The radical derivatives of oxygen (O2) are the most important free radicals in the biological systems. The activated O2 intermediates together with secondarily formed radicals like hydroxyl radicals are able to destroy membrane lipids, proteins, DNA, and cartilage (Ramprasath et al., 2005). Neutrophils, macrophages and dendritic cells generate ROS in large amounts in response to activation in FCAinduced rats (Arulmozhi et al., 2011). Moreover, ROS produced by macrophages, lymphocytes and endothelial cells contribute to the destruction of cartilage (Mythilypriya et al., 2007). Increased lipid peroxidation and decreased enzymatic and non-enzymatic antioxidants are found in RA patients, the MDA levels in RA patients are significantly higher than normal, but the activities of GSH-Px and SOD are lower. There was an increase in oxidative stress and a low antioxidant status in patients with RA (Xiao et al., 2011). These findings suggest that oxidant stress plays a very important role in the pathogenesis of RA disease. Our study also showed that levels of two oxidation products, MDA and NO, in AIA rats were higher than those in the control, and meanwhile, the activities of antioxidants were lower (Fig. 6). These results in AIA rats were consistent with the RA patients. The results also demonstrated that the antioxidative activity of FA may be one of anti-RA mechanisms. RA is part of Bi Zheng in the theory of traditional Chinese medicine, which is characteristic of a syndrome marked by arthralgia and dyskinesia of the joints and limbs because of an attack of the meridians by wind, dampness, and heat or cold pathogens (Lam et al., 2008). RA's marked symptoms of hemorheological disorders, which included the decrease of blood flow

velocity and disturbance of the normal blood flow, were consistent with Bi Zheng (Li et al., 2009). As blood is a non-Newtonian fluid, blood viscosity increases as shear rate decreases. Blood cells have the characteristic of deformability, aggregation and adhesion, and these rheology parameters also have shear dependence. WBV at a low shear rate is mainly determined by the red blood cell aggregation. If red blood cell aggregates break up, then the apparent viscosity of the blood falls with the increase of shear rate. In a high shear rate condition, WBV is mainly determined by the deformability of the erythrocyte (Windberger et al., 2003; Shin et al., 2004; Wells, Merrill 1961). Our results showed that the WBV of the model group was the highest at all shear rates. FA reduced WBV, and the relative mechanism might work by depressing the aggregation of erythrocytes, which might help prevent microthrombus formation (Wang et al., 1999). Antioxidant activity and hemorheology-modulatory activity of FA contribute to the treatment of RA in rats. The linear correlations between antioxidant activities and WBV at different rates were calculated in order to analyze the relationship between the two potential mechanisms. The level of NO had a strong direct correlation with high shear rates, whereas the activity of GSH-Px had great inverse correlation with high shear rates. The results indicate that NO and GSH-Px have a close relationship with WBV. These findings may become an effective way of evaluating RA disease. In summary, FA showed significant therapeutic effects on Freund's adjuvant-induced arthritis in rats without obvious adverse effects. This anti-RA effect may be attributed to antioxidant and hemorheology modulative activities. The FA may have a potential clinical utility in patients with rheumatoid arthritis.

Acknowledgments This work was supported in part by a Grant (BK20131309) from the Natural Science Foundation of Jiangsu Province and the Tang Center for Herbal Medicine Research.

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