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Anti-rheumatoid arthritic activity of flavonoids from Daphne genkwa.

ABSTRACT

The aim of the study was to investigate the anti-rheumatoid arthritic activity of four flavonoids from Daphne genkwa (FFD) in vivo and in vitro. Flavonoids of D. genkwa were extracted by refluxing with ethanol and purified by polyamide resin. An in vivo carrageenan-induced paw edema model, tampon-granuloma model and Freund's complete adjuvant (FCA)-induced arthritis mouse model were used to evaluate the anti-rheumatoid arthritic activities of FFD. Moreover, nitric oxide (NO) release and neutral red uptake (NRU) in lipopolysaccharide (LPS)-induced murine macrophage RAW264.7 cells were used to evaluate the anti-inflammatory effect in vitro. In addition, antioxidant effect of FFD was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. A high dose of FFD significantly reduced the degree of acute inflammatory paw edema in mice as a response to carrageenan administration (p < 0.01). FFD displayed a dose-dependent inhibition of granuloma formation in mice (p < 0.05). FFD also inhibited chronic inflammation in adjuvant-induced arthritis rats when administered orally at the dose of 50 mg/kg/day (p < 0.001). In addition, FFD suppressed the production of NO and exhibited immunoregulatory function in LPS-activated RAW264.7 cells in a dose-related manner. Simultaneously, FFD revealed conspicuous antioxidant activity with [IC.sub.50] values of 18.20 [micro]g/ml. FFD possesses significant anti-inflammatory and antioxidant activity, which could be a potential therapeutic agent for chronic inflammatory disorders such as rheumatoid arthritis.

Published by Elsevier GmbH.

Keywords:

Daphne genkwa

Flavonoids

Rheumatoid arthritis

Adjuvant-induced arthritis

RAW264.7 cells

Introduction

Rheumatoid arthritis (RA), a chronic autoimmune disease, is characterized by cartilage loss, synovial hyperplasia and joint damage, mainly in the ankles (Islander et al. 2011). Adjuvant arthritis (AA) in rats shares many pathologic characteristics with RA suffered in humans, such as extremities swelling, synovium hyperplasia and cartilage degradation. Thus, the resemblance between RA and AA makes it reasonable to screen new drugs for the treatment of RA (Jawed et al. 2010). However, numerous molecules, such as IL-1, IL-6 and TNF-[alpha], prostaglandins (PG) and nitric oxide (NO), underlie local tissue destruction during the process of RA (Gallin and Snyderman 1999). As part of our research for anti-rheumatoid arthritic activity, we selected NO, which may react with superoxide resulting in peroxynitrite formation and cell toxicity (Huang et al. 2012), to examine whether FFD had potential effects against inflammatory response in LPS-induced RAW246.7 cells. On the contrary, it is commonly accepted that free radicals, acting as mediators, stimulate or promote inflammatory processes, while their neutralization by antioxidants can decrease inflammatory symptoms (Delaporte et al. 2002). Thus, recent studies in screening of active anti-inflammatory agents were more focused on antioxidants (Perry et al. 1999).

The treatment goals for this disease are long-term relief of pain, prevention of joint inflammation and suppression of pannus formation and morphological changes (Li et al. 2012). Drugs used for the treatment of RA suppress biological processes that contribute to the onset and symptoms of inflammation (Sondhi et al. 2002). In fact, the recent clinical therapeutic medicines, such as glucocorticosteroid, non-steroidal anti-inflammatory drugs, immunosuppressant and so on, have serious side effects including gastrointestinal injury, renal irritations, and cardiovascular risks (Rainsford 2007). Instead, natural products are expected to treat RA due to lower toxicity and fewer side effects (Zhang et al. 2007).

Daphne genkwa Sieb. et Zuce. (Thymelaeaceae), a well-known traditional Chinese medicine, has long been used for diuretic, anti-tussive, expectorant and anticancer purposes (Jiang su New Medical College 1985). In recent years, the pharmacological activity of this plant, including anticancerous (Zhan et al. 2005; Zhang et al. 2006; Zheng et al. 2007a, 2007b), anti-inflammatory (He et al. 2008) and immunoregulation (Gao et al. 2006), have been published. However, the detailed anti-chronic inflammatory activity of D. genkwa Sieb. et Zucc, especially on RA, has not been reported so far. Additionally, the cellular action mechanisms for the modulation of the inflammatory response of this plant have not been elucidated. Therefore, we investigated the effects of FFD on RA and the mechanisms of inflammation. Moreover, DXM, proved to be one of the strongest and most widely used corticosteroid anti-inflammatory drug (Kraaij et al. 2011), was selected as the positive control drug of western medicine. TCP, successfully used as an agent in the treatment of RA in China (Zhang and Sun 2010), was chosen as the positive control drug of traditional Chinese medicine. The anti-rheumatoid arthritic activity of FFD was detected in vivo and in vitro compared with above-mentioned control medicines.

Materials and methods

Plant materials

D. genkwa Sieb. Et Zucc. (Thymelaeaceae) was purchased from Anqing, Anhui Province of China, and authenticated by Professor Ping Li, Department of Traditional Chinese Medicine, China Pharmaceutical University. Voucher specimens were deposited in the State Key Laboratory of Natural Products and Functions, China Pharmaceutical University.

Animals

Rats and mice in either sex, weighing 150-200 g and 20-25 g, respectively, were purchased from the Experimental Animal Center of Zhejiang Province. All of them were kept in an animal light-control house (12 h light/dark cycle), with food and tap water ad libitum before experimentation.

Preparation of FFD

The air-dried D. genkwa Sieb. Et Zucc (2 kg) was extracted three times with ethanol under reflux at 100[degrees]C. The extracting solution was chromatographed on a polyamide column, and then eluted step gradients. The obtained fraction was evaporated to dryness and the extract was kept in desiccator for experiments.

HPLC analysis

FFDs were analyzed using an 1100 series HPLC (Agilent Technologies, USA) equipped with a UV detector (338 nm). An Agilent SB-[C.sub.18] analytical column (250 mm x 4.6 mm, 5 [micro]m) was used at the flow rate of 1.0 ml/min. The mobile phase consisted of 70% methanol/30% [H.sub.2]O with 0.1% formic acid.

Acute toxicity assay

For the toxicity assay, mice were randomly divided into control and test groups (n = 10). The test groups were treated with increasing doses of 0.1, 0.5, 2.5 and 12.5 g/kg of FFD, respectively. The toxic symptoms and mortality rates were observed daily and continuously for two weeks after intragastric administration.

Carrageenan-induced paw edema in mice

This method was carried out as described by Calhoun et al. (1987). Mice were randomly assigned to seven groups (n = 10) for further experiments. Doses of 40, 80 and 160 mg/kg of FFD were intragastrically administered in different groups. Reference groups received 0.8447 mg/kg of DXM (Beijing Double-Crane Pharmaceutical Co., Ltd., China) and 225.25 mg/kg of TGP (provided), normal group and control group received 0.5% CMC-Na solution. 1 h after pretreatment, edema was induced by subcutaneous injection 0.02 ml of 1% (w/v) carrageenan (Sigma-Aldrich, USA) diluted into saline in the right hind paw. The paw thickness was measured immediately prior to carrageenan injection and at 1, 2, 4 and 6h after the administration of the edematogenic agent using a spiral micrometer (Shengke Instrument Co., Ltd., China).

Cotton pellet granuloma

The method was described in the previous study (Ismail et al. 1997). Sterile cotton pellets (10 [+ or -] 0.5 mg) were implanted subcutaneously in the groin of mice anesthetized by 3% chloral hydrate. Then the stylolitic wounds were coated in gentamicin (Sigma-Aldrich, USA) to protect against infection. The animals were given 40, 80, 160 mg/kg of FFD, DXM (0.8447 mg/kg), TGP (225.25 mg/kg) or saline (10 ml/kg) orally in succession daily for seven days. On the eighth day, the mice were sacrificed, and the cotton pellets were removed and dried overnight at 60[degrees]C to determine the dried weights. Results were expressed as

Inhibition ratio (%) = ([M.sub.t] - [M.sub.0]) control - ([M.sub.t] - [M.sub.0]) treated/ ([M.sub.t] - [M.sub.0]) control x 100

where [M.sub.t] was the cotton weight after implantation and [M.sub.0] was the cotton weight before implantation.

Adjuvant-induced chronic arthritis

According to the method mentioned by Newbould (1963), 0.1 ml of FCA (1 mg heat-killed Mycobacterium butyricum (Sigma-Aldrich, USA) desiccated in 100 [micro]l liquid paraffin) was injected subdermally into the right hind metatarsal footpad of rats, the same volume of paraffin oil alone was given to rats in normal group. On the nineteenth day after injection, the control group (n = 8) was given a 0.5% CMC-Na solution. The others (n = 8) were administrated with FFD (12.5, 25, and 50 mg/kg), DXM (0.58 mg/kg) or TGP (156.25 mg/kg) by intragastric administration once daily from the nineteenth day to the thirty-fifth day. The paw volumes were measured using a plethysmometer.

The arthritic scores were estimated from day 25 to 30 by means of a visual scoring system, which was performed on a 0-4 scale as follows; 0 (normal), 1 (mild swelling or erythema), 2 (moderate swelling and erythema), 3 (severe swelling and erythema), 4 (excess swelling with joint rigidity).

Finally, rats were sacrificed. The weights of body and organs (spleen, thymus, liver and adrenal gland) were recorded.

To describe tibial inflammation intuitively, the tibial bones from rats were removed for histological examination. Paws were fixed in 10% phosphate buffered formalin for 24 h. After being dehydrated and embedded in paraffin, H&E-stained slices reflected the histological pathological changes of foot.

Cell culture

The murine macrophage RAW264.7 cells, obtained from the pharmacological research center of China Pharmaceutical University, were cultured in DMEM (Linz, Austria) supplemented with 10% FBS (Sigma-Aldrich, USA) and 100U/ml of penicillin (Betastar, USA) and 100U/ml of streptomycin (Betastar, USA) at 37[degrees]C in an atmosphere of 5% C02 and subcultured every 3 days.

MTT cell viability assay

Based on the pre-test, 0.25-5 [micro]g/ml of LPS (Sigma-Aldrich, USA) was available to test. RAW264.7 cells were seeded in 96-well plate (1 x 105/well, Corstar[R] Corporation, USA) and incubated for 24 h. Cells were pretreated with several concentrations of FFD, DXM or TGP for 1 h and then co-stimulated with 0.5 |xg/ml of LPS for 24 h. After that, the supernatant was carefully discarded, 180 p.1 DMEM and 20 [micro]l of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, USA) were added and consecutively incubated for another 4 h. The formazan crystals, converted from MTT by mitochondrial succinate dehydrogenase in live cells, were dissolved in DMSO (Linz, Austria), and the absorbance was measured at 570 nm using a microplate reader (Awareness, USA). All experiments were performed in triplicate.

Measurement of nitric oxide (NO) production

Cells were preprocessed with different concentrations of FFD, DXM or TGP for 1 h. Subsequently, cellular NO production was induced by adding 0.5 [micro]g/ml of LPS and incubation at 37[degrees]C for 24 h. Then, NO from the supernatant was measured using nitric oxide assay kit (SunBio, North Korea). All experiments were performed in triplicate.

Neutral red uptake test (NRU)

Phagocytic capacity of the macrophage was assessed by NRU. Briefly after measurement of NO production, the supernatant was removed from each well and replaced with neutral red working solution, following incubation for 30 min. Then, they were rinsed 3 times using D-Hank's buffer salt. The supernatant was removed and the cells were solubilized in 0.01 M ethanol/acetic acid solution. The absorbance of the final solution was measured at 550 nm using microplate reader. All experiments were performed in triplicate.

DPPH assay

Free radical scavenging activity of FFD was determined by DPPH method (Wang et al. 1998). Briefly, methanol solutions of samples were mixed with DPPH solution (0.4 mmol/1, Sigma-Aldrich, USA). Concentrations of FFD, TGP and DXM in reaction solutions were 10, 20, 40, 60, 80, and 100 |xg/ml. The reaction mixtures were shaken vigorously and incubated for 30 min in the dark, and the absorbance of each sample was measured at 517 nm against a blank using a spectrophotometer. The scavenging capability of DPPH radical was calculated by the following equation:

Inhibition (%)= [([A.sub.blank] - [A.sub.sample])/[A.sub.blank]] x 100

where [A.sub.blank] and [A.sub.sample] were the absorbances of blank and sample, respectively. [IC.sub.50] was defined as the concentration that scavenges 50% of DPPH free radical activity. Ascorbic acid (i.e. Vc) acted as a positive control for this study.

Statistical analysis

The data were expressed as mean [+ or -] S.E. Statistical analysis was performed with one-way ANOVA followed by a Tukey's post hoc test for multiple comparisons tests.

Results

Acute toxicity of FFD

The safety of FFD was evaluated using acute toxicity assay. The doses of 0.1-12.5 g/kg of FFD did not exhibit any mortality or associated symptoms, such as convulsion, ataxia, diarrhea and so on, during the next 14 days. These indicated that FFD had low toxicity profile.

Quantitative determination of flavonoids in FFD

The chromatogram of FFD is shown in Fig. 1. FFD in D. genkwa Sieb. Et Zucc. is luteolin, apigenin, hydroxygenkwanin and genkwanin, which together make up 92% of the purified fraction.

Anti-rheumatoid arthritic activity in vivo

Effects of FFD on carrageenan-induced paw edema

As seen in Fig. 2, subcutaneous injections of 1% carrageenan induced an edema, reaching its maximum at 2 h and remaining to 6 h. Compared to the low doses of FFD (40 and 80 mg/kg), high dose (160 mg/kg) acutely decreased the thickness of hind paw from 2 to 6 h (p < 0.01). During this period, pre-treatment with DXM and TGP also potently contributed to inhibit carrageenan-stimulated hind paw edema (p < 0.001 and p < 0.01, respectively).

Cotton pellet induced granuloma

The results shown in Fig. 3 found that FFD displayed dose-dependent inhibition of the cotton pellet granuloma. 160 mg/kg of FFD significantly inhibited granuloma tissue with a value of 31.96%, a slightly lower than 37.11% obtained from the positive control DXM (p<0.05). However, FFD (40 and 80 mg/kg) reduced its formation by 12.37% and 17.53% in comparison to the negative control group, the same degree as inhibition obtained from TGP (225.25 mg/kg).

Effect of FFD on contralateral paw volume

Fig. 4A shows paw volume development of the contralateral hind limb of normal and arthritic rats after the onset of the inflammatory process. The hind limb of control rats started swelling after subplantar FCA administration. Rats showed notable and dose-dependant attenuation in contralateral paw volume after treatment with FFD (12.5,25 and 50 mg/kg) on the thirty-fifth day. Moreover, the amelioration of DXM was equally potent as the effects of TGP and FFD (50 mg/kg), statistically (p < 0.001). Also, the inhibitions of FFD (12.5 and 25 mg/kg) were significant (p < 0.01), just inferior to the positive control drugs.

Effect of FFD on arthritic scores

Animals in the control group presented with severe arthritic lesions, the extent is shown by Fig. 4B. Rats treated with FFD (50 mg/ml) shared a significant reduction in arthritic scores with DXM in comparison with rats treated with FCA alone at day 30 and 35 (p < 0.001). Also, rats treated with FFD (25 mg/ml) had less severe arthritis than those in control group on the same days (p<0.01). There was a decrease in arthritic scores in rats treated with low-dose FFD and TGP as compared to control rats. However, the decrease was not statistically significant.

Effect of FFD on organ index

The results are shown in Fig. 4C. There was a significant increase in liver index and spleen index (p<0.01). Whereas a decrease in thymus index (p<0.05) of control rats as compared to normal can be seen. Treatment with FFD (25 mg/kg) showed a significant decrease in liver index as compared to control (p<0.05). Rats given FFD (50 mg/kg) pronouncedly decreased spleen index (p<0.01), and this value obtained by DXM group even more decreased as compared to control (p< 0.001). When compared with the control, animals treated with FFD (50 mg/kg) markedly increased thymus index (p<0.05), but significantly decreased in DXM group (p< 0.001). However, there were no significant differences in adrenal gland index among all groups.

Effect of FFD on histochemistry of inflamed joint

Photomicrographs of the sections revealed that the inflamed joint of AA can be seen in Fig. 4D. Rats in the control group exhibited massive accumulation of mono or poly-morphonuclear cells and synovial hyperplaisia (Fig. 4D, b). Histological observation of normal group exhibited absence of necrosis phenomenon (Fig. 4D, a). Although FFD (25 mg/kg) and FFD (50 mg/kg) exhibited slight synovial hyperplasia, they both ameliorated inflammatory cells infiltration and thickening of synovial membrane (Fig. 4D, c and d). Treatment with DXM showed a lower degree of lesions, similar to normal (Fig. 4D, e). However, there were apparent deformations and thickening of synovial cells in the group of TGP (Fig. 4D, f).

Anti-rheumatoid arthritic activity in vitro

Effect of FFD on cell viability of RAW246.7 cells

Effect of FFD on cell viability of RAW246.7 cells was evaluated to establish the appropriate concentration range for the analysis of NO production and NRU. As expected, it showed no significant differences between the cells treated with LPS alone and FFD (2.5-50 |xg/ml), neither did DXM and TGP (Fig. 5A). Therefore, these concentrations were feasible to further investigation.

Effect of FFD on NO production

Pre-incubation of various concentration of FFD, DXM and TGP on RAW246.7 cells was operated to examine whether they could reverse LPS-induced accumulation of NO production (Fig. 5B). NO release significantly increased after induction with LPS, as compared to the normal (p < 0.001). With all doses of FFD, DXM and TGP, we observed a dose-dependent inhibition in NO relative value. Cells treated with FFD, DXM and TGP remarkably reversed the production of NO as concentrations in the range of 10-50 [micro]g/ml, 50-200 [micro]g/ml and 200-500 [micro]g/ml, respectively.

Effect of FFD on immunoloregulation

The effect of FFD on immunoloregulation was assessed by neutral red assay. As seen in Fig. 5C, cells induced by LPS resulted in abnormal increase in neutral red uptake as compared to normal (p < 0.05). FFD (25 and 50 [micro]g/ml) and DXM (50,100 and 200 [micro]g/ml) displayed significant decrease in uptake of neutral red, which modified the phagocytic capacity in comparison to those in the control. Whereas it was found to be non-significant statistically with all doses of TGP.

Free radical scavenging activity of FFD

DPPH free radical scavenging activity of FFD was shown in Fig. 6. The antioxidant activities of various concentrations of FFD, TGP and Vc in the dose range of 10-100 [micro]g/ml were dose-dependently. The value of Vc at the 100 [micro]g/ml was 96.22% with [IC.sub.50] of 13.07 [micro]g/ml. Compared to Vc, FFD exhibited similarly radical scavenging activity, which was 95.50% at 100 [micro]g/ml and with [IC.sub.50] of 18.20 [micro]g/ml. However, the relative lower radical scavenging activity was observed by TGP ([IC.sub.50] = 32.09 [micro]g/ml).

Discussion

Rheumatoid arthritis (RA) is characterized by cartilage loss and synovial inflammation (Islander et al. 2011). Persistent and chronic inflammation in the synovial membrane is the pathological basis of RA (Zhang and Dai 2012). The direct anti-inflammatory effects of FFD were evaluated in acute, subacute and chronic inflammatory models. In our investigation, FFD exerted rapid onset, potent and sustained action, which indicated anti-rheumatoid arthritis potentiality.

Carrageenan-induced edema is a typical model used to assess anti-inflammatory effect of trial drugs (Yi et al. 2010). It is considered to be biphasic, the primary acute phase (0-2 h) is correlated with the release of histamine, serotonin, kinins and some mediators. However, the secondary chronic phase (over 2-5 h) is relevant to bradykinin, leukotrienes, polymorphonuclear cells, prostaglandins and cyclooxygenase products (Antonio and Souza Brito 1998; Perianayagam et al. 2006; Deciga-Campos et al. 2007). Leukotrienes, generated from lipoxygenase, are sensitive to steroidal anti-inflammatory drugs, such as DXM (Blackwell et al. 1980). In our study, the inhibition, shown by FFD (160 mg/kg) in carrageenan-induced paw edema, decreased sharply after 2 h, which was quite similar to that of DXM. Therefore, the inhibition by FFD was probably due to lipoxygenase for suppression of leukotrienes synthesis, supporting that natural products were commonly to aim directly at the tumescence in phase two (Calixto et al. 2004).

Granulation tissue, which is rich in capillaries, is produced by capillary endothelial cells and fibroblasts (Zhang and Dai 2012). The inflammatory granuloma is a typical feature to establish chronic inflammatory response of organism (Ismail et al. 1997). Cotton-induced granulation hyperplasia has been widely used to evaluate the transudative, exudative and subacute inflammation (Purnima et al. 2010). The dry weight of the cotton correlated well with the hyperplasia of granulomatous tissue (Swingle and Shideman 1972). Herein, the inhibiting effects of FFD on this nonspecific inflammation were evaluated by cotton-induced granuloma. The results showed that FFD inhibited granuloma formation in a dose-dependent manner. The inhibition of FFD (160 mg/kg) was the same degree as positive control DXM, suggesting that FFD efficiently suppressed the subacute inflammation process.

Rat adjuvant arthritis is a widely used classic model, which shares many similar characteristics to RA in human, including histology and immunology (Jawed et al. 2010). FCA is mainly responsible for stimulating cell-mediated immunity to increase production of certain immunoglobulins (Zhang and Dai 2012). Injections of FCA induced primary and secondary chronic arthritis (Walz et al. 1971). During the development of adjuvant arthritis, a large amount of pro-inflammatory agent may cause inflammation at multiple sites (Taurog et al. 1988). This kind of polyarthritis initiates a secondary stage of the inflammatory response and induces the lesions in the paws on days 21 and 25 (Noguchi et al. 2005). Furthermore, the onset of pain associated with swelling of the limbs and disability of joint function attributes to release of various inflammatory mediators such as macrophage colony-stimulating factor (MCSF), cytokines (1L-1B and TNF[alpha]) and so on (Eric and Lawrence, 1996). Cytokines can create a positive feedback mechanism between fibroblast- and macrophage-like synoviocytes associated with RA (Sweeney and Firestein 2004). Activation of TNF[alpha] triggers a series of signaling events, including stimulation of transcription factor NF-[kappa]B and the MAP kinase family (Chen and Goeddel, 2002). In our investigation, FFD mainly consisted of luteolin, apigenin, genkwanin and hydroxygenkwanin. We focused on the four flavonoids because of their semblable structure (Fig. 1). It is reported that apigenin and luteolin significantly inhibited TNF[alpha]-induced NF-[kappa]B transcriptional activation (Funakoshi-Tago et al. 2011). Treatment with FFD significantly decreased swelling of contralateral paw, which might be related to production of cytokines and activation of NF-[kappa]B.

Liver, spleen, adrenal gland and thymus are vital organs involved in immune responses. Niu et al. (2011) suggested that lymphoid organs played an important role in treatment of RA. Subplantar administration of FCA significantly increased spleen weights, decreased thymus weights and altered hepatic function compared to saline-injected animals (Ismail et al. 2008). Thymus atrophic action, via the hypothalamo-pituitary-adrenal axis suppression, is considered as a systemic adverse effect of steroidal anti-inflammatory drugs (Dujovne and Azarnoff 1975). Significant atrophy of thymus and spleen as compared to normal might be due to side effects of DXM. However, treatment with FFD (50 mg/kg) decreased spleen index and liver index, whereas increased thymus index, displaying immunoregulatory effects.

Various pathological conditions, such as cellular injury, inflammation and autoimmune diseases, are related to NO (Singh et al. 2000). When treated with LPS in RAW264.7, some intracellular signaling pathways are activated, likewise, many genes encoding inflammatory mediators are induced, including inducible nitrite oxidative synthase (iNOS) (Surh et al. 2001). NO, produced by iNOS, is chiefly involved in enhancing flammatory responses (Rimai et al. 2005). Thus, NO inhibitors are a necessary deterrent of inflammatory process (Lee et al. 2009). The present study showed that FFD (10-50 [micro]g/ml) was significantly suppressed NO production in a concentration-dependent manner. That two upstream DNA regions of the iNOS promoter is involved in transcription activation, such as NF-[kappa]B binding sites, IFN-[gamma] response elements and TFN-[gamma]-activated factor binding (Mizutani et al. 1998). The monomeric flavonoids decreased NFKB-dependent gene expression indicating TNF-[alpha] secretion and NO production might be partially mediated by the inhibition of NF-[kappa]B (Park et al. 2000). Fortunately, it is reported that the known compounds in FFD, apigenin and luteolin, significantly inhibited TNF[alpha]-induced NF-[kappa]B transcriptional activation (Funakoshi-Tago et al. 2011). Meanwhile, another two ingredients shared a familiar structure with them. These evidences demonstrated that FFD might suppress NO production by blocking transcriptional activation of NF-[kappa]B, serving as modulators of immune response in RAW264.7.

In conclusion, a high dose of FFD has anti-inflammatory effects on various models. It can not only inhibit destruction of joint structure but also improve health status in adjuvant arthritis and ameliorate clinical symptoms, such as paw edema, granulation tissue hyperplasia and so on. In particular, the underlying mechanisms of anti-inflammatory effects possibly correlate with free radical scavenging, down-regulation of pro-inflammatory molecules including leukotriens and cytokines, and responses of NF-[kappa]B. These findings suggest FFD has potential to overcome serious side effects of synthetic agents for long-term anti-rheumatoid arthritis.

ARTICLE INFO

Article history:

Received 20 September 2013

Received in revised form 31 October 2013

Accepted 26 January 2014

Acknowledgment

This work was supported by funds of the Nature Science Foundation of Jiangsu Province (Grant No. BK20131309).

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Cui-Ping Jiang (a), Xin He (b), Xiao-Lin Yang (c), Su-Li Zhang (a), Hui Li (a), Zi-Jing Song (a), Chun-Feng Zhang (a,d), *, Zhong-Lin Yang (a), Ping Li (a), Chong-Zhi Wang (d), Chun-Su Yuan (d)

(a) State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China

(b) School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210000, China

(c) Guangdong Provincial Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, China

(d) Tang Center of Herbal Medicine Research, and Department of Anesthesia and Critical care. University of Chicago, Chicago, IL 60637, USA

Abbreviations: FFD, four flavonoids from Daphne genkwa: FCA, Freund's complete adjuvant; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NO, nitric oxide; NRU, neutral red uptake; LPS, lipopolysaccharide; DPPH, 2,2diphenyl-1-picrylhydrazyl; DXM, dexamethasone; TGP, total glucosides of paeony; RA, rheumatoid arthritis; AA, adjuvant arthritis; PG, prostaglandins; DMEM, Dulbecco's Modified Eagle's Medium; iNOS, nitrite oxidative synthase.

E-mail addresses: zhangchunfeng67@163.com, ggyx536@163.com (C.-F. Zhang).

http://dx.doi.org/10.1016/j.phymed.2014.01.009

* Corresponding author at: State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjia Lane, Nanjing, Jiangsu 210009, China. Tel.: +86 25 86185129.
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Author:Jiang, Cui-Ping; He, Xin; Yang, Xiao-Lin; Zhang, Su-Li; Li, Hui; Song, Zi-Jing; Zhang, Chun-Feng; Ya
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:May 15, 2014
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