Therapeutic effects of standardized Vitex negundo seeds extract on complete Freund's adjuvant induced arthritis in rats.

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ARTICLE IN PRESS Phytomedicine xxx (2014) xxx–xxx

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Therapeutic effects of standardized Vitex negundo seeds extract on complete Freund’s adjuvant induced arthritis in rats Cheng-Jian Zheng a,1 , Xiang-Xiang Zhao a,1 , Hong-Wei Ai a , Bing Lin a , Ting Han a , Yi-Ping Jiang a , Xin Xing b,∗∗ , Lu-Ping Qin a,c,∗ a

Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China Department of Plastic Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, PR China c Shanghai Key Laboratory for Pharmaceutical Metabolite Research, Shanghai 200433, PR China b

a r t i c l e

i n f o

Article history: Received 16 August 2013 Received in revised form 5 December 2013 Accepted 14 February 2014 Keywords: Vitex negundo Adjuvant arthritis Rheumatoid arthritis Anti-arthritis

a b s t r a c t The seeds of Vitex negundo L. (Verbenaceae) have been commonly used as a folk remedy for the treatment of rheumatism and joint inflammation in Traditional Chinese Medicine. This study aimed to evaluate the anti-arthritic activity of the extract of V. negundo seeds (EVNS) using Freund’s complete adjuvant (CFA) induced arthritis (AA) in rat model. As a result, EVNS, with abundant phenylnaphthalene-type lignans, significantly inhibited the paw edema, decreased the arthritis score and spleen index, and reversed the weight loss of CFA-injected rats. Histopathological studies showed a marked decrease of synovial inflammatory infiltration and synovial lining hyperplasia in the joints of EVNS-treated animals. The remarkable decrement of serum inflammatory factors (TNF-␣, IL-1␤ and IL-6) were observed in EVNS-treated rats, whereas, IL-10, an anti-inflammatory cytokine, was found to be significantly increased by EVNS. The expressions of COX-2 and 5-LOX in PBMC were also inhibited by administration of EVNS. Our results demonstrated that V. negundo seeds possessed potential therapeutic effect on adjuvant induced arthritis in rats by decreasing the levels of TNF-␣, IL-1␤ and IL-6 and increasing that of IL-10 in serum as well as down-regulating the levels of COX-2 and 5-LOX, and therefore may be an effective cure for the treatment of human rheumatoid arthritis. © 2014 Elsevier GmbH. All rights reserved.

Introduction Rheumatoid arthritis (RA) is one of the most chronic destructive diseases to human health, which is also called “immortal cancer”, characterized by inflammatory cell infiltration and proliferation of synovial tissue, accompanied by bone destruction (Imboden 2009; Pincus and Callahan 1993). It can rapidly progress into multisystem inflammation with irreversible joint damage thus causing premature mortality, disability and compromised quality of life in the industrialized and developing world (Silman and Hochberg 2001; Brooks 2006). Nowadays, disease-modifying antirheumatic drugs (DMARDs) supplemented with non-steroidal anti-inflammatory drugs

∗ Corresponding author at: Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China. Tel.: +86 21 81871300; fax: +86 21 81871300. ∗∗ Corresponding author. E-mail addresses: [email protected] (X. Xing), [email protected] (L.-P. Qin). 1 These authors contributed equally to this work.

(NSAIDs), steroid hormone and biologics (TNF-␣ antibody and the decoy TNF-␣ receptor, etc.) remains the major strategy in the treatment of RA (Silman and Hochberg 2001; Scott et al. 1998). According to the guidelines of the American College of Rheumatology, newly diagnosed RA patients were strongly recommended to begin treatment with NSAIDs for relieving noceceptive pain and controlling inflammation, with combined use of DMARDs for reducing disease activity, preventing joint deformity and improving joint function (Doan and Massarotti 2005). However, administration of these drugs is associated with severe adverse effects, including gastrointestinal lesions, cardiovascular complications, reproductive toxicity, and etc. (Couzin 2004; Kremers et al. 2004). Therefore, more and more attention has been paid to plant-derived anti-RA drugs with high efficacy and few side effects. Recent investigations have estimated that 60–90% of RA patients are very likely to use botanicals (Wang et al. 2012). This growing interest in alternative medical practices clearly indicates the need for more safe and effective anti-RA botanicals used in the traditional medicine. Vitex negundo L. (Verbenaceae), an important medicinal plant in the Vitex species, has been used as reputed herbal medicine with versatile pharmacological activities in China, India, Japan,

http://dx.doi.org/10.1016/j.phymed.2014.02.003 0944-7113/© 2014 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Zheng, C.-J., et al., Therapeutic effects of standardized Vitex negundo seeds extract on complete Freund’s adjuvant induced arthritis in rats. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.02.003


ARTICLE IN PRESS C.-J. Zheng et al. / Phytomedicine xxx (2014) xxx–xxx

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Indonesia, East Africa and South America (Zheng et al. 2009a). The seeds of this plant are commonly used as a condiment for edible purpose (Kunkel 1984) and have been widely used in folk medicine for anti-inflammatory (Zheng et al. 2009a, 2010; Chawla et al. 1992), analgesic (Zheng et al. 2009b), and antioxidant purposes (Ono et al. 2004), containing abundance of bioactive lignans (Zheng et al. 2009a; Ono et al. 2004), terpenoids (Chawla et al. 1992; Zheng et al. 2010; Ono et al. 2004) and flavonoids (Chawla et al. 1991; Bhargava 1989). In Traditional Chinese Medicine, V. negundo seeds were commonly used for a series of inflammation related disorders, such as rheumatism, chronic bronchitis, chronic gastritis and colitis (Vimal et al. 2011). In Ayurvedic Medicine, V. negundo seeds also found a good reputation for the treatment of rheumatoid arthritis (Mohiuddin et al. 1977). In our previous studies, we also demonstrated the significant antinociceptive and antiinflammatory activities of the extract of V. negundo seeds (EVNS) in mice in a dose-dependent manner (Zheng et al. 2009b). The traditional use of V. negundo seeds as an anti-inflammatory agent, together with modern pharmacological studies, suggested it has potential therapeutic effects in the treatment of RA, although no previous studies have focused on the in vivo anti-RA activity of this medicinal seed. Therefore, the present study was conducted to investigate the efficacy of the extract of V. negundo seeds (EVNS) as anti-arthritic agents and explore its potential mechanism on adjuvant-induced arthritis (AA) in rats. Materials and methods Chemicals and solvents Complete Freund’s adjuvant (CFA) and Histopaque 1083 were purchased from Sigma Chemical Co., USA. Methotrexate was bought from Shanghai Sine Pharmaceutical Co., Ltd., China. ELISA kits of TNF-␣, IL-1␤, IL-6, and IL-10 were purchased from R&D system, USA, while COX-2 and 5-LOX were bought from MyBioSource, USA. All the other chemicals and biochemical used were of the highest grade available. Plant material The seeds of V. negundo (Chinese name ‘Huang-Jing-Zi’) were obtained from Wanglang National Nature Reserve, Sichuan Province, and were identified by Professor Lu-ping Qin, Second Military Medical University. A voucher specimen (#2006-168) has been deposited in the herbarium of the Department of Pharmacognosy, School of Pharmacy, Second Military Medical University. Preparation of extract The preparation of the extract of V. negundo seeds (EVNS) was conducted according to the method described by Zheng et al. (2009b). Briefly, the air-dried and powdered seeds were extracted with 80% EtOH three times (3×) for 2 h each time. After removal of the solvent under reduced pressure, the residue was evaporated to dryness. The dry residue was suspended in water with 1% w/v sodium carboxyl methyl cellulose (Na-CMC) for pharmacological studies. The doses employed are expressed as mg of the dried extract per kg body weight. HPLC analysis of the extract of V. negundo seeds (EVNS) The extract of V. negundo seeds (EVNS) was dissolved in 50% methanol prior to HPLC analysis. This was performed on an Agilent1100 system with a Zorbax Extend-C18 chromatographic column (250 mm × 4.6 mm, 5 ␮m) at 30 ◦ C with a H2 O (+0.1% HCOOH) (A)/acetonitrile (B) gradient, a sample injection volume of 20 ␮l, flow rate of 0.8 ml/min, and a detection wavelength 254 nm.

Samples were analyzed by using a gradient program as follows: run was commenced with 8% B, linear gradient to 15% B within 14 min, followed by linear gradient to 25% B in 21 min, and finally linear gradient to 55% B in 35 min. Animals Male Wistar rats weighing 160–180 g body weight were procured from the Experimental Animal Center of Second Military Medical University (Shanghai, China). They were kept in an environment with controlled temperature (24–26 ◦ C) and photoperiod (12:12 h) light–dark cycle. Animals were given standard commercial rat chow and water ad libitum and processed following the internationally approved ethical guideline of the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals, and the experiments were carried out with the approval of the Animal Experimentation Ethics Committee of the Second Military Medical University (approval number: 20130214). Adjuvant induction Complete Freund’s adjuvant (CFA, sigma) was prepared by suspending heat-killed BCG in liquid paraffin at 10 mg/ml. Each rat, except the normal control group, was injected intradermally with 0.1 ml of Freund’s complete adjuvant (Sigma product) into the left hind metatarsal footpad of rat for induced inflammation (Sindhu et al. 2011). The animals were divided into five groups of six animals in each as follows: • Group I – Normal control • Group II – Rats with adjuvant-induced arthritis (AA) • Group III – AA + methotrexate (3 mg/kg, twice a week, orally by gastric intubation) • Group IV – AA + EVNS (340 mg/kg/day, orally by gastric intubation) • Group V – AA + EVNS (85 mg/kg/day, orally by gastric intubation). The normal group and AA model group were given an equal volume of the vehicle (CMC-Na) at the same time. During the course of the experiment, the body weight of rats was measured every 7 days. And the rats were assessed every three days for signs of arthritis between days 1 and 28. Each paw was recorded on an ordinal scale as follows: 0 = unaffected, 1 = 1 type of joint affected, 2 = 2 types of joints affected, 3 = 3 types of joints affected, 4 = 3 types of joints affected and maximal erythema and swelling (Kokkola et al. 2003). The maximum arthritic score per rat was set at 8 (4 points × 2 hind paws). The rat paw edema was measured on days 0, 7, 14 and 21 expressed as the foot-breadth of ankle by vernier caliper. On the 28th day, the rats were sacrificed by decapitation. Thymus and spleen were dissected out, washed in ice-cold saline, patted dry and weighed (Sundaram et al. 2011). The indices of thymus and spleen were expressed as the ratio (mg/g) of thymus and spleen wet weight versus body weight, respectively (Zhang et al. 2004). Index of thymus and spleen assay At day 28 after immunization, the rats were sacrificed via anesthesia (pentobarbital sodium, 40 mg/kg, i.p.). The thymus and spleen were then promptly removed and weighed. The index of thymus and spleen were expressed as the ratio of thymus and spleen wet weight versus body weight (mg/g), respectively (Zhang et al. 2004). Isolation of peripheral blood mononuclear cells (PBMC) The experiment was carried out according to the modified method of Radhika et al. (2007). Briefly, 3 ml Histopaque 1083




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Fig. 1. HPLC analysis of the extract of V. negundo seeds (EVNS). (A) HPLC–UV chromatogram of standard reference; (B) HPLC–UV chromatogram of the sample used in the experiment. Peaks were detected at 254 nm (1, vitexdoin A; 2, 6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-3,4-dihydro-2-naphthaldehyde; 3, vitedoin A; 4, vitedoamine A).

solution was added to a 15 ml centrifuge tube and then 3 ml blood was layered on top of this density gradient and centrifuged for 30 min at 1500 rpm at room temperature. The upper white layer consisting of mononuclear cells was decanted and washed with phosphate buffered saline (PBS), which was repeated twice. After that the pellet was resuspended in PBS-Tween and subjected to freeze-thaw cycle three times. The resulting lysate was used as the enzyme source. Determination of serum TNF-˛, IL-1ˇ, IL-6 and IL-10 Rat blood was obtained and allowed to clot for 1 h at room temperature. Serum was recovered and frozen at −20 ◦ C until assayed (Anderson et al. 1996). Serums TNF-␣ concentrations were quantified by ELISA according to the manufacturer’s protocol and all samples assayed for TNF-␣ were measured using the test kit individually. The same procedure was used to assay IL-1␤, IL-6 and IL-10. Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (LOX) level assay in PBMC The expression levels of COX-2 and 5-LOX in PBMC were analyzed using an ELISA immunoassay according to the manufacturers’ instructions (MyBioSource, USA). Briefly, the COX level assay utilizes the peroxidase component of cyclooxygenases.

The peroxidase activity was assayed colorimetrically by monitoring the appearance of oxidized N,N,N ,N -tetramethyl-pphenylenediamine (TMPD) at 450 nm (Sindhu et al. 2011). Similar procedure was used to assay 5-LOX, with the substitution of antibodies specific to this enzyme. Histopathological studies On day 28, all rats were sacrificed via anesthesia after serum collection. Hind paws and knee joints were removed from the rats for histological examination (n = 6). For histological evaluation, we performed hematoxylin and eosin (H&E) staining of tissue specimens of joints, fixed in 10% neutral buffered formalin and decalcified in EDTA for 30 days at 4 ◦ C. After procession for paraffin embedding, sections were cut at 3 ␮m thickness, stained with hematoxylin–eosin and viewed under a light microscope for histopathological changes (Connor et al. 1995). Statistical analysis The results were analyzed using SPSS 13.0 statistical software. One-way ANOVA was used for determining the statistically significant differences between the values of various experimental groups. Data were expressed as mean ± SD and a p < 0.05 was considered statistically significant.




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Fig. 2. Effect of EVNS on the paw edema of CFA-injected rats. Values are expressed as mean ± SD (n = 6) in each group. EVNS at the dose of 340 and 85 mg/kg/day showed significant reduction in paw edema swelling when compared with the AA group (*p < 0.05, **p < 0.01).

Results HPLC analysis of EVNS In order to elucidate the chemical profile of V. negundo seeds, we performed the HPLC analysis of its ethanol extract (EVNS). Our previous phytochemical studies on this medicinal seed revealed the abundance of phenylnaphthalene-type lignans (Zheng et al. 2009a,b). Based on those compounds we have already obtained from V. negundo seeds, four major components in the HPLC fingerprint of EVNS were identified and recognized as vitexdoin A (Rt: 22.319 min), 6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)3-hydroxymethyl-7-methoxy-3,4-dihydro-2-naphthaldehyde (Rt: 33.802 min), vitedoin A (Rt: 36.587 min), and vitedoamine A (Rt: 39.800 min) (Chawla et al., 1991; Greish et al. 2012), the contents of which were calculated as 1.22, 3.74, 3.26, and 0.22 mg/g (in crude drug), respectively (Fig. 1).

Effects of EVNS on the paw swelling, arthritis score and weight growth CFA-injected animals exhibited a marked peripheral edema of the injected paw, subsequently observable 24 h after the injection. Administration of EVNS at both 340 and 85 mg/kg significantly (p < 0.05) reduced the paw edema from day 14th when compared with the model group rats. MTX (3 mg/kg) also exhibited notable efficacy in inhibiting the paw edema of the arthritic rats. By the 21st day of treatment, the paw edema in EVNS and MTX treated animals has almost disappeared (Fig. 2). An evident reduction in the clinical arthritis score was also observed in the group treated with EVNS at both 85 and 340 mg/kg, which was significant throughout the treatment period from the 14th day. The potent efficacy of MTX (3 mg/kg) was also observed from day 14 to 28 (Fig. 3). Animals treated with CFA gained weight more slowly than normal control group and these arthritic rats showed a marked weight loss (p < 0.05) compared with the vehicle control from the third week (Fig. 4). Our study showed the weight loss of CFA-injected rats could be reversed by administration of EVNS at both 340 and 85 mg/kg. Although the rate of weight gain in EVNS-treated rats was lower than it had been initially, it was still much higher than that of CFA-injected rats. When compared with the control, animals treated with EVNS did not show significant weight loss during the experiment.

Fig. 3. Effect of EVNS on the arthritis score of CFA-injected rats. Values are expressed as mean ± SD (n = 6) in each group. EVNS at the dose of 340 and 85 mg/kg/day showed significantly decreasing of arthritic score from day 9 to 28 when compared with the AA group (*p < 0.05, **p < 0.01).

Effects of EVNS on the index of thymus and spleen As shown in Fig. 5, the index of thymus and spleen in AA group were markedly increased, compared with the normal group. The index of spleen of the drug treated animals was significantly decreased and the level was brought back to near normal in either MTX (3 mg/kg) or EVNS (85 and 340 mg/kg) groups. MTX (3 mg/kg) also decreased the index of thymus of the arthritic rats, whereas no significant impact on the index of thymus was observed in EVNS treated animals. Histopathological examination H and E stained sections of the hind limbs of the normal rats showed intact articular cartilage with normal joint space and synovial tissue (Fig. 6, Group I). Sections from the AA model animals showed synovial lining hyperplasia and massive mononuclear cell infiltration of the synovial tissue. Severe bone destruction was also visible in the sections (Fig. 6, Group IIA and B). The MTX group revealed a marked decrease of synovial inflammatory cell infiltrate and synovial lining hyperplasia with moderate obliteration of the joint cavity (Fig. 6, Group III). The group treated with EVNS (340 mg/kg/day) exhibited significant reduction of synovial lining hyperplasia and synovial inflammatory cell infiltrate compared to the AA model group. Also the normal form and structure of joint cartilage were kept and observed in the same section (Fig. 6, Group IV). Sections from the EVNS (85 mg/kg/day) treated animals showed

Fig. 4. Effect of EVNS on the weight growth of CFA-injected rats. Values are expressed as mean ± SD (n = 6). The AA model rats showed a marked weight loss when compared with the normal group (*p < 0.05, **p < 0.01), whereas the EVNS treated groups did not show significant weight loss.




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Fig. 5. Effects of EVNS on the indices of thymus and spleen. Values are expressed as mean ± SD (n = 6) in each group. The index of thymus and spleen index in AA group were markedly increased when compared with the normal group (## p < 0.01). EVNS at the dose of 340 and 85 mg/kg/day significantly decreased the level of index of spleen but no significant impact on the index of thymus when compared with the AA group (*p < 0.05, **p < 0.01).

evidence of irregular joint surface but normal synovial tissue (Fig. 6, Group V). Histopathological studies showed the evidence of EVNS in suppressing chronic inflammation, pannus formation and bone destruction of AA in rats. Effect of EVNS on adjuvant-induced expression of TNF-˛, IL-1ˇ, IL-6 and IL-10 in serum There was a significant increase of TNF-␣, IL-1␤ and IL-6 in AA model rats, while the level of IL-10 was declined, compared to the normal rats. Both MTX (3 mg/kg) and EVNS (85 and 340 mg/kg) showed potent inhibitory effects on the production of TNF-␣ and IL-1␤ (Fig. 7). Furthermore, EVNS treatment (85 and 340 mg/kg) markedly increased the production of IL-10 in the serum of AA rats (p < 0.05). The same effect was also observed in MTX (3 mg/kg) treated group. In addition, the level of IL-6 was significantly decreased in MTX-treated group, while only high dose of EVNStreated rats (340 mg/kg) showed significant reduction of IL-6. Effects of EVNS on the expression of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (LOX) The level of COX-2 and 5-LOX in peripheral blood mononuclear cells (PBMC) were significantly increased in adjuvant induced rats when compared with the normal group. Administration of EVNS (85 and 340 mg/kg) significantly decreased the level of COX-2 and 5-LOX, compared with AA group (Fig. 8). Discussion Rheumatoid arthritis (RA) is a systemic inflammatory disease which manifests itself in multiple joints of the body with chronic polyarthritis (Silman and Hochberg 2001). As a chronic autoimmune disease, RA is characterized by cytokine-mediated inflammation of the synovial lining of the joints, leading to erosions of the cartilage and bone and sometimes joint deformity (Wong et al. 2010). Pain, stiffness, swelling, deformity and, eventually, loss of function of the joints are common manifestations in RA (Soeken et al. 2003). Allopathic medications prescribed to alleviate symptoms of this disease mainly include pain killers (analgesics), NSAIDs, DMARDs and corticosteroids (steroids) (Patwardhan et al. 2010), which however have limitations due to their severe adverse effects. Herbal medicines, with high efficacy and few side effects,

therefore become more and more popular and well explored in recent decades and constitute a potentially important avenue leading to novel therapeutic agents for RA (Otari et al. 2010). Vitex negundo is an important medicinal plant, commonly used in China TCM and India Ayurvedic medicine for the treatment of various inflammation related disorders. Especially, its seeds are widely used for rheumatism and inflammation of joints (Chawla et al., 1992). Our study was therefore conducted to investigate the in vivo anti-arthritic activity of V. negundo seeds in detail. CFA-induced arthritis in rats is a widely used experimental animal model of inflammatory polyarthritis characterized by infiltration of the synovial membrane and associated with joints destruction, which resembles closely to those clinical and pathological features of human RA (Shinde et al. 1999; Patil et al. 2011; Narendhirakannan and Limmy, 2012). Rats with adjuvant-induced arthritis (AA) are therefore widely accepted as an appropriate model for screening potential anti-RA drugs (Greish et al. 2012). Paw swelling and arthritic scores are indices of measuring the antiarthritic activity of various tested drugs and employed here to evaluate the activity of EVNS at doses of 85 and 340 mg/kg. Our study revealed that EVNS administered groups showed marked reduction in paw volume and significant decrease in arthritic scores when compared with the AA model group. The observed inhibitory effects were in a dose-dependent manner. In addition, the significant weight loss in CFA-induced rats was reversed by administration of EVNS. In the present study, EVNS-treated rats continued to show normal weight gain. Since inflammatory cytokines (such as TNF-␣) levels have been reported to be associated with weight loss in various human diseases (Tracey et al. 1989), the anti-inflammatory activity of EVNS can be responsible for its reversal of weight loss in CFA-induced rats. Thymus and spleen are two major organs of the body’s immune system, of which the relative weights are commonly used as the preliminary indicators to evaluate the immunoregulatory activity the tested drugs. The spleen normally filters out and destroys old and damaged blood cells and removes antigens by phagocytosis, which plays a key role in preventing infection by producing white blood cells called lymphocytes and acting as a first line of defense against invading pathogens (Handy et al. 2002). Similar arguments apply to the thymus, which normally processes immature precursor T-lymphocytes (derived from bone marrow and initially located in the outer cortex of the thymus) into the mature immunocompetent T-cells of the medulla (Vos and Van Loveren, 1998). Our results clearly revealed that EVNS reversed splenomegaly of rats induced


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Fig. 6. Effects of EVNS on histopathological changes. Group I – normal rats (H&E stain 50×), Group II (A, B) – arthritis, which shows severe synovial lining hyperplasia and cell infiltration (H&E stain 50×), Group III – AA + methotrexate (H&E stain 50×), Group IV – AA + EVNS (340 mg/kg/day) (H&E stain 50×), Group V – AA + EVNS (85 mg/kg/day) (H&E stain 50 × ).

by CFA injection but exhibited no effect on the weight loss of thymus, which suggested that EVNS played an important role in the immune system mainly by inhibiting the enlargement of the spleen and the systemic expansion of immune cells.

RA mainly targets synovium and joints as the primary sites of the inflammatory process, which can be intuitively reflected by histopathological assays. The histology of joints from CFAimmunized rats in our study showed severe proliferation of the




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Fig. 7. Effect of EVNS on adjuvant-induced expression of TNF-␣, IL-1␤, IL-6 and IL-10 in serum. Values are expressed as mean ± SD (n = 6) in each group. There was a significant increase of TNF-␣, IL-1␤ and IL-6 in AA model rats, while the level of IL-10 was declined, compared to the normal rats (## p < 0.01). EVNS (85 and 340 mg/kg) showed potent inhibitory effects on the production of TNF-␣ and IL-1␤. Furthermore, EVNS treatment (85 and 340 mg/kg) markedly increased the production of IL-10 in the serum of AA rats in a dose-dependent manner when compared with the AA group (*p < 0.05, **p < 0.01).

synovium, with significant infiltration of inflammatory cells, cartilage damage and bone erosion. Our results revealed EVNS can not only suppress inflammatory response, but also attenuate bone destruction in AA rats, thus may be one optimal strategy in the treatment of RA.

As the major effectors of synovitis, macrophages act via cytokines secretion, mainly including TNF-␣, IL-1␤ and IL-6 (Boissier et al. 2012). TNF-␣ plays a crucial role in joint inflammation and destruction of cartilage and bone of RA patients (Feldmann et al. 1996), which can regulate the production of pro-inflammatory

Fig. 8. Expression of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (LOX) in rat PBMC. Values are expressed as mean ± SD (n = 6) in each group. The levels of COX-2 and 5-LOX in peripheral blood mononuclear cells (PBMC) were significantly increased in adjuvant induced rats when compared with the normal group (## p < 0.01). Administration of EVNS (85 and 340 mg/kg) significantly decreased the level of COX-2 and 5-LOX, compared with AA group (*p < 0.05, **p < 0.01).




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cytokines including IL-1 and IL-6 in vitro (Carteron 2000) and stimulate synoviocytes to mediate synovial hyperplasia and produce extracellular matrix-degradative enzymes and chemokines facilitating the progress of the arthritic erosion (McInnes and Schett, 2007). IL-1␤ is a pro-inflammatory cytokine driving joint inflammation as well as systemic signs of inflammation, playing a vital role in the pathogenesis of RA. IL-6, one of the most important physiopathological factors of bone resorption contributes mainly in an unspecific manner to the synthesis of autoantibodies, such as rheumatoid factor (RF) (Álvarez 2009). IL-10, a potent antiinflammatory cytokine primarily produced both by monocytes and Th2 (Bozkurt et al. 2006), has been regarded as an upstream regulator that controls the progression of RA synovitis negatively (Apparailly et al. 1998; Joosten et al. 1999; Walmsley et al. 2005). As shown in Fig. 6, the levels of TNF-␣, IL-1␤ and IL-6 raised significantly in AA model group. Administration of EVNS markedly attenuated TNF-␣, IL-1␤ and IL-6 expression in serum and increased the level of IL-10, suggesting the mechanism for the beneficial effect of EVNS on RA may involve both down-regulating the level of TNF-␣, IL-1␤ and IL-6 and up-regulating the expression of IL-10. Prostaglandins are important lipid mediators that are produced at high levels in inflamed tissues such as rheumatoid synovium (Bombardieri et al. 1981; Davies et al. 1984). They are products of the cyclooxygenase (COX) pathway of arachidonic acid metabolism. COX-2 is the product of an immediate early response gene in inflammatory cells and responsible for the enhanced production of prostaglandins. 5-LOX is the key enzyme involved in the synthesis of leukotrienes from arachidonic acid (Sindhu et al. 2011). High levels of COX-2 and 5-LOX were expressed in PBMC of AA model group, while significant decrease was observed in EVNS treated groups (Fig. 8), which suggests that inhibition of arachidonic acid metabolism may be another mechanism of EVNS’s anti-arthritic action. By HPLC fingerprint analysis, we found that the main components of EVNS were phenylnaphthalene-type lignans. Since phenylnaphthalene-type lignans were proved to be effective as analgesic and anti-inflammatory agents (Zheng et al. 2009a,b), we suggested that the total lignans in V. negundo seeds can be responsible for the observed anti-arthritic activity in the present study, which merits further studies regarding the precise site and the mechanism of action. In conclusion, our study showed that EVNS, with abundant phenylnaphthalene-type lignans, was effective on CFA-induced arthritis in rats, as evidenced by both signs and pathology scores. The anti-arthritic activity of EVNS observed can be attributed to its ability in down-regulating the levels of TNF-␣, IL-1␤ and IL-6 and up-regulating IL-10 concentration as well as decreasing the levels of inflammatory enzymes COX-2 and 5-LOX, thus suggesting V. negundo seeds as a potent strategy for the treatment or prevention of RA, which merits further studies as regards to fully elucidate the anti-RA constituents and mechanisms of action.

Conflicts of interest The authors disclose that there are no conflicts of interest.

Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 81102773; U1203202), Outstanding Youth Program of Shanghai Medical System (XYQ2013100) and TCM Modernization Foundation of Science and Technology Commission of Shanghai (No. 10DZ1972000).

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Therapeutic Effects of Acetone Extract of Saraca asoca Seeds on Rats with Adjuvant-Induced Arthritis via Attenuating Inflammatory Responses.

Effect of oral administration of ethanolic extract of Vitex negundo on thioacetamide-induced nephrotoxicity in rats.

Chemical investigation and anti-inflammatory activity of Vitex negundo seeds.

Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund׳s adjuvant induced arthritis in rats.

A new triterpenoid glycoside from Vitex negundo.

Protective effect of Asarum extract in rats with adjuvant arthritis.

Effect of galantamine on adjuvant-induced arthritis in rats.

Therapeutic effects of PADRE-BAFF autovaccine on rat adjuvant arthritis.

Therapeutic effects of dichloromethane fraction of Securidaca inappendiculata on adjuvant-induced arthritis in rat.

Antibacterial behaviour of Vitex negundo extract assisted ZnO nanoparticles against pathogenic bacteria.

The effects of a minimally invasive laser needle system on complete Freund's adjuvant-induced arthritis.

Antiarthritic activity of a polyherbal formulation against Freund's complete adjuvant induced arthritis in Female Wistar rats.

Oral Administration of Shark Type II Collagen Suppresses Complete Freund's Adjuvant-Induced Rheumatoid Arthritis in Rats.

Effects and mechanisms of Geniposide on rats with adjuvant arthritis.

Xylopia aethiopica (Annonaceae) fruit extract suppresses Freund's adjuvant-induced arthritis in Sprague-Dawley rats.

Antioxidant effects of Genkwa flos flavonoids on Freund׳s adjuvant-induced rheumatoid arthritis in rats.

Therapeutic effects of total steroid saponin extracts from the rhizome of Dioscorea zingiberensis C.H.Wright in Freund's complete adjuvant induced arthritis in rats.

Insight into the bronchodilator activity of Vitex negundo.

Effects of cichoric acid extract from Echinacea purpurea on collagen-induced arthritis in rats.

Therapeutic Effect of Saponin Rich Fraction of Achyranthes aspera Linn. on Adjuvant-Induced Arthritis in Sprague-Dawley Rats.

Prophylactic effect of aqueous extract of Sesamum indicum seeds on ethanol-induced toxicity in male rats.

Protective effects of the standardized extract of Zingiber officinale on myocardium against isoproterenol-induced biochemical and histopathological alterations in rats.

Vitamin D metabolism in rats with adjuvant-induced arthritis.

Cyclic AMP production during adjuvant-induced arthritis in rats [proceedings].