Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats Bing Lin a,1, Ping Han b,1, Wei Yue a, Xue-Qing Ma c, Khalid Rahman d, Cheng-Jian Zheng a, Lu-Ping Qin a,n, Ting Han a,nn a
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, PR China Center for Disease Control and Prevention, Jinan Military Region, PLA, Ji'nan 250014, PR China c Department of Pharmaceutical analysis, School of Pharmacy, NingXia Medical University, 1160 Shenli Street, Yinchuan 750004, PR China d Faculty of Science, School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 July 2013 Received in revised form 7 May 2014 Accepted 18 May 2014
Ethnopharmacological relevance: Xanthium strumarium L. fruit (Xanthiu fruit) has been traditionally used as a medicinal herb in China for the treatment of many ailments including rheumatoid arthritis. However, the anti-arthritic activity of Xanthium strumarium fruit has still not been demonstrated. In the present study, we confirmed that the extract of Xanthium strumarium (EXS) prevents rheumatoid arthritis induced by Complete Freund's Adjuvant (CFA) in rats. Materials and methods: Male Wistar rats (1607 10 g) were immunized by intradermal injection of 0.1 mL of CFA into the left hind metatarsal footpad. EXS was administered orally at a dose of 300 and 75 mg/kg once a day after the induction of adjuvant arthritis. Methotrexate (3 mg/kg, twice a week) was used as a positive control. Paw swelling, arthritic score, body weight loss, spleen index, thymus index, serum cytokines, inflammatory mediators and histological change were measured. The chemical profile of EXS was analyzed by HPLC-DAD. Results: We found that the EXS significantly suppressed paw swelling and arthritic score, increased body weight loss and decreased the thymus index The overproduction of TNF-α and IL-1β was remarkably suppressed in the serum of all EXS-treated rats, and in contrast IL-10 was markedly increased. The level of COX-2 and 5-LOX was also decreased with EXS treatment. Ten phenolic acid derivatives were identified from 14 detected peaks by HPLC-DAD with the reference substances and verified by LC–MS. Conclusions: These results suggest the potential effect of EXS as an anti-arthritis agent towards CFAinduced arthritis in rats. Xanthium strumarium has the potential to be regarded as a candidate for use in general therapeutics and as an immune-modulatory medicine in rheumatoid arthritis. & 2014 Published by Elsevier Ireland Ltd.
Keywords: Xanthium strumarium L. Adjuvant arthritis Inflammation Anti-arthritis
1. Introduction Rheumatoid arthritis (RA) is a chronic destructive disease of the joints and is characterized by chronic proliferative synovitis, infiltration of inflammatory cells into the synovial tissue of joints, and cartilage destruction. It can rapidly progress into a multisystem inflammation with irreversible joint destruction, thus increasing the risk of mortality (Harris, 1990; Praveen and Suchita, 2013). Over 60 million adults in the world wide are affected by this disease and its prevalence is three times more in
n
Corresponding author. Tel./fax: þ 86 21 81871300. Corresponding author. Tel./fax: þ86 21 81871306. E-mail addresses: [email protected] (L.-P. Qin), [email protected] (T. Han). 1 Bing Lin and Ping Han equally contributed to this manuscript; both should be considered as first author. nn
women than men. The age of onset is between late twenties and early fifties, however, no age is immune to the disease (Winters and Geissman, 1969). The life span of patients with RA is shortened due to an increase in cardiovascular and cerebrovascular diseases (Wolfe et al., 1994). The non-steroidal anti-inflammatory drugs (NSAIDs) and biologics (e.g., TNF-α antibody and the decoy TNF-α receptor) represent a prominent group of drugs used in the treatment of RA. However, the usage of these drugs is associated with severe adverse effects, including gastrointestinal bleeding and cardiovascular complications (Winters and Geissman, 1969). Therefore, more and more patients are experimenting with medical botany options in order to cope with this debilitating disease. A recent investigation has estimated that 60–90% of patients with rheumatoid arthritis are very likely to use botanicals (Rao et al., 1999). The genus Xanthium (Family Asteraceae) is represented by 25 species in the world, of which three species and one variety
http://dx.doi.org/10.1016/j.jep.2014.05.023 0378-8741/& 2014 Published by Elsevier Ireland Ltd.
Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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are found abundantly throughout China (Han et al., 2007). Historically, Xanthium strumarium L., an important medicinal plant in the Xanthium species, has been used as reputed herbal medicines in China, Europe, Indo-China, Malaysia and America (Kamboj and Saluja, 2010). The whole plant is used as a medicine for curing nasal sinusitis, headache, urticaria and arthritis. It has also been reported to possess curative effects against chronic bronchitis, chronic rhinitis, allergic rhinitis, lumbago and other ailments (Zhu, 1998) and is used by various native American tribes to relieve constipation, diarrhea and vomiting (Kamboj and Saluja, 2010). An infusion of the plant has also been used in the treatment of rheumatism, diseased kidneys and tuberculosis (Chopra and Nayar, 1986). Additionally, Xanthium strumarium tissues contain high amounts of bioactive compounds such as glycosides, phytosterols, phenolic acids and xanthiazones, and the extract of this herb exhibit analgesic, antibacterial, antifungal, antimalarial, antirheumatic, antispasmodic, antitussive, cytotoxic, hypoglycemic and stomachic properties (Cumanda et al., 1991; Mahmoud, 1998; Qin et al., 2006; Winters and Geissman, 1969). In our previous studies of croton-oil-induced ear edema test and acetic acidinduced writhing test, it was shown that oral administration of EXS to mouse can attenuate inflammatory ear edema as well as reducing the number of writhing induced by acetic acid in mice in a dose-dependent manner (Han et al., 2007). The traditional use of Xanthium strumarium as an anti-inflammatory agent may have potential anti-arthritic effects in the treatment of RA, although as far as we know, no studies have focused on the in vivo effects of this herb. Therefore, the current study was designed to confirm the anti-arthritic effect and explore the potential mechanism of Xanthium strumarium on adjuvant-induced arthritis (AA) in rats, an animal model of rheumatoid arthritis.
water with 1% w/v sodium carboxyl methylcellulose (Na-CMC). The doses employed are expressed as mg of dried extract per kg body weight. 2.4. HPLC-DAD analysis of EXS An Agilent 1100 series HPLC-DAD system comprising a vacuum degasser, binary pump, autosampler, thermostated column compartment and DAD (Agilent, USA) was used for acquiring chromatograms and UV spectra. A Zorbax SB C-18 reversed-phase column (250 mm 4.6 mm, 5 μm) together with a Phenomenex C-18 guard column (10 mm 4.6 mm, 5 μm) were used with column temperature set at 25 1C. The mobile phase consisted of 0.5% (v/v) phosphoric acid in water (A) and methanol (B). Separation was achieved by a linear gradient elution from 20–40% B in 0–20 min, 40–50% B in 20–50 min and 50% B in 50–60 min at a flow rate of 0.8 mL/min. The DAD detector wavelength was set at 327 nm for acquiring chromatograms. Ultra-violet (UV) spectra were acquired from 200 to 700 nm. EXS prepared as mentioned above. Ten reference substances were used for the qualitative analysis: 1,3,5-O-tricaffeoylquinic acid, 1-O-caffeoylquinic acid, 1,4-Odicaffeoylquinic acid, 3-O-caffeoylquinic acid, chlorogenic acid, 4-O-caffeoylquinic acid, 1,3-O-dicaffeoylquinic acid and 1,4-Odicaffeoylquinic acid were isolated and purified from the fruits of Xanthium strumarium in our laboratory; 1,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid and 3,4,5-O-tricaffeoylquinic acid were purchased from National Institute for the Control of Drug and Biological Products (Beijing, China). The compounds were identified or deducted based on comparing individual peak retention times with the reference substances and verified by LC–MS (Han et al., 2009). 2.5. Animals
2. Materials and methods 2.1. Reagents and apparatus Agilent 1100 Series High Performance Liquid ChromatographyDiode Array Detector (HPLC-DAD) (Agilent, USA), Plethysmometer 7140 (UGO BASILE, Italy), FLx800 Fluorescence Microplate Reader (Bio-Tek, USA); Histopaque 1083 and Complete Freund's adjuvant (CFA) were purchased from Sigma (Sigma Chemical Co., USA), Methotrexate (Shanghai Sine Pharmaceutical Co., Ltd., China). IL-1β, TNF-α, IL-6 and IL-10 ELISA kits were purchased from the R&D system (R&D system, USA), COX-2 and 5-LOX ELISA kits were obtained from Biocalvin (Biocalvin, Suzhou, China). All the other chemicals and biochemical used were of the highest grade available.
Male Wistar rats, aged 6–8 weeks (160 710 g), were purchased from the Experimental Animal Center of Second Military Medical University (Shanghai, China). All rats were allowed to acclimatize for 1 week before the experiments were started. The rats were housed under standard laboratory conditions (room temperature 2572 1C, relative humidity 40–70% and free access to water) maintained on a 12-h light 12-h dark cycle. This experiment was approved by the Bioethics Committee of the Second Military Medical University (Shanghai, China), and the procedures of the experiment strictly adhered to generally accepted international rules and regulations. 2.6. Induction of adjuvant arthritis and treatment
The ripe fruits of Xanthium strumarium, Xanthii Fructus were purchased from an Herbal Medicinal Materials Company of Anton pharmaceutical Co., Ltd. (Bozhou, Anhui province, China), and authenticated by Prof. H.C. Zheng (The Second Military Medical University, Republic of China). A voucher specimen has been deposited in School of Pharmacy, Second Military Medical University, Shanghai, China.
Complete Freund's Adjuvant (CFA, sigma) was prepared by suspending heat-killed BCG in liquid paraffin at 10 mg/mL. The Wistar rats were immunized by intradermal injection of 0.1 mL of CFA into the left hind metatarsal footpad of rat (Perera et al., 2011). The rats were divided into 5 groups randomly, in which the rats with AA received EXS at 300 and 75 mg/kg once daily by intragastric (i.g.) from day 2. Methotrexate (3 mg/kg, twice a week) was used as a positive control. The normal and control group rats were given an equal volume of vehicle (1% CMC-Na) at the same time.
2.3. Preparation of plant extract
2.7. Assessment of arthritis
Dried fruits of Xanthii Fructus (5 kg) were extracted with 75% ethanol at 70 1C for 3 times (2 h each time). The extract was then combined and evaporated to dryness under the reduced pressure (60 1C), and yielded 300 g residue (Extract of Xanthium strumarium fruits, EXS). For pharmacological studies, EXS was suspended in
The volume of the hind paw swelling was measured with electronic water plethysmographicall before first immunization (basic value, day 0) and repeat on days 7, 14, 21 and 28. Meanwhile the body weight of rats was measured every 7 days, and the changes in body weight are shown as weight growth (g). Arthritis
2.2. Plant material
Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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score was used to evaluate the clinical severity of AA rats. A wellestablished, widely used scoring system was used to evaluate the severity of AA (Zhang et al., 2009). Paws were examined and graded for severity and loci of erythema, swelling and induration using a 5-point scale: 0 ¼no signs of disease, 1¼ signs involving the ankle/wrist, 2¼ signs involving the ankle plus tarsal of the hind paw and/or wrist plus carpals of the forepaw, 3 ¼signs extending to the metatarsals or metacarpals, and 4 ¼severe disease involving the entire hind or fore paw. The maximum arthritic score per rat was set at 8 (4 points 2 hind paws).
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Diluent were added into the wells of a microtiter plate coated with rat COX-2 antibody, and then 100 μL HRP-conjugate reagent was added and incubated for 1 h at 37 1C. After washing with Wash Solution (400 μL) for 4 times, chromogen solution A and B each 50 μL were added and incubated for 15 min at 37 1C, protected from light. Then, added Stop Solution 50 μL and the optical density were calculated with an automated Colter microplate reader at 450 nm. The same procedure was used to assay 5-LOX, with the substitution of antibodies specific for this enzyme. 2.11. Measurement of serum cytokines concentrations
2.8. Thymus index and spleen index assay The thymus and spleen were promptly removed and weighed when the animals sacrificed at day 28 after immunization (Pentobarbital Sodium, 40 mg/kg; i.p.). The thymus index and spleen index were expressed as the ratio (mg/g) of thymus and spleen wet weight versus body weight, respectively (Zhang et al., 2004). 2.9. Isolation of peripheral blood mononuclear cells (PBMC) The method of isolation of peripheral blood mononuclear cells was modified from Radhika et al. (2007). In brief, Histopaque 1083 solutions (3 mL) were placed in a 15 mL tube and 3 mL blood was layered on top of this density gradient. The blood cells were separated into two fractions after centrifugation 1500 rpm for 30 min: the mononuclear cells were in the upper white layer plus the majority of platelets at the interface region, and the erythrocytes and granulocytes were in the lower layer. The plasma layer on top with no cells was removed and discarded. The monocytes from the buffy coat were carefully taken off by aspiration and washed with phosphate buffered saline (PBS). The pellet was resuspended in PBS-Tween and subjected to freeze-thaw cycle three times and the resulting lysate was used as the enzyme source. 2.10. Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) level in PBMC
When the blood was standing for 30 min, the serum was collected by centrifugation at 3000 rpm for 10 min and stored at 20 1C prior to analysis. The concentrations of cytokines such as TNF-α, IL-1β, IL-6 and IL-10 were quantified by radioimmunoassay according to the manufacturer's protocol. All samples assayed for TNF-α were measured using the test kit individually. The same procedure was used to assay IL-6, IL-1β and IL-10, with the substitution of antibodies specific to the cytokines. 2.12. Histological changes When the rats were sacrificed via anesthesia after serum collected on day 28. Knee joints and hind paws were removed from the rats for histological analysis. The joints were fixed in 10% phosphate buffered formalin, decalcified in 10% EDTA for 30 days at 4 1C, and then embedded in paraffin. Serial paraffin sections (5 mm) were stained with hematoxylin and eosin (H&E). 2.13. Statistical analysis The data represent the mean 7S.D. (n ¼ 8). Differences between experimental groups were tested using one-way ANOVA; p values less than 0.05 were considered significant.
3. Results 3.1. HPLC-DAD qualitative analysis
The COX-2 expression level in PBMC was determined using an ELISA immunoassay kit according to the manufacturer's instructions (Biocalvin). In brief, the PBMC lysate 10 μL and 40 μL Sample
The HPLC-DAD analysis of EXS (Fig. 1) shown that over 14 peaks were detected within 60 min, among which 10 compounds were
Fig. 1. HPLC-DAD analysis of EXS. HPLC-DAD was used for the qualitative analysis of the main constituents of the EXS. A total of 14 peaks were detected in the EXS, and 10 compounds were deduced based on comparing individual peak retention times with those of the authentic reference substances and verified by LC–MS. There were (1) 1-Ocaffeoylquinic acid; (2) unknown; (3) 3-O-caffeoylquinic acid; (4) chlorogenic acid; (5) 4-O-caffeoylquinic acid; (6) 1,3-O-dicaffeoylquinic; (7) unknown; (8) unknown; (9) 1,4-O-dicaffeoylquinic acid; (10) 1,5-O-dicaffeoylquinic acid; (11) 4,5-O-dicaffeoylquinic acid; (12) 1,3,5-O-tricaffeoylquinic acid; (13) 3,4,5-O-tricaffeoylquinic acid; and (14) unknown.
Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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identified or deducted based on comparing individual peak retention times with those of the authentic reference substances and verified by LC–MS. They were (1) 1-O-caffeoylquinic acid, (3) 3-Ocaffeoylquinic acid, (4) chlorogenic acid, (5) 4-O-caffeoylquinic acid, (6) 1,3-O-dicaffeoylquinic acid, (9) 1,4-O-dicaffeoylquinic acid, (10) 1,5-O-dicaffeoylquinic acid, (11) 4,5-O-dicaffeoylquinic acid, (12) 1,3,5-O-tricaffeoylquinic acid and (13) 3,4,5-Otricaffeoylquinic acid. 3.2. Effects of EXS on paw swelling, arthritis score and weight change Obvious joint swelling was shown in all immunized rats after 24 h. An initial development in the manifestations of inflammation from day 1 to day 4 was observed, and then it showed a brief decrease in the inflammatory signs from day 5 to day 6. This phase
was followed by a quick reappearance of inflammation on day 11 and day 12. Significant increasing of paw volume and arthritic scores were observed in CFA immunized rats when compared with the normal control rats. However, the polyarthritis symptoms and hind paw swelling reduced after treatment with EXS and MTX on day 14, 21 and 28. Overall, the reduced paw volume after treatment with both concentrations of EXS and MTX showed no significant differences from the normal group, while the level was significantly (po0.01) lower than the control group, the same results were observed on day 21 and day 28 (Fig. 2). All rats showed a significant increase of arthritic scores upon induction by CFA before day 10, however the rats treated with either EXS (both concentrations) or MTX successfully reduced the arthritic scores in rats compared with the control rats significantly from day 10 to day 28 (Fig. 3). The relationship between the extent of joint inflammation and the weight loss were investigated. As shown in Fig. 4, in the second week after adjuvant injection, the control group rats showed a significant weight loss (p o0.05), followed by normal weight gain in the subsequent weeks, whereas the EXS and MTX treatment groups did not show significant weight loss. 3.3. Effects of EXS on thymus index and spleen index
Fig. 2. Effects of EXS on paw swelling in rats. 40 male Wistar rats (160 7 10 g, 8/group) were divided into 5 groups randomly and immunized by intradermal injection of 0.1 mL of CFA into the left hind metatarsal footpad except normal group. The MTX group received methotrexate twice a week at 3 mg/kg as a positive control. The EXS group (300, 75 mg/kg) was received EXS once daily by intragastric (i.g.) from day 2. The volumes of the hind paw swelling (mL) were measured before first immunization (basic value, day 0) and repeat on days 7, 14, 21 and 28. The increment of paw swelling calculates as follows: Δ (mL) ¼volume day 14 (21 or 28) – volume day 0; Data represent the mean 7S.D. (n¼ 8). Values are statistically significant at np o 0.05, nnp o 0. 01 compared with the control group.
The thymus index and spleen index are associated with immunological functions. The thymus index and spleen index were assayed when the rats sacrificed at day 28, and the results showed that thymus index and spleen index of the control group were increased compared with normal group. Both concentrations of EXS (300, 75 mg/kg) showed a marked decreased of thymus index. However, there was no significant effect of spleen index in both concentrations of EXS treatment (Fig. 5). Both thymus index and spleen index were decreased in the MTX treatment group. 3.4. Effects of EXS on inflammatory mediators COX-2 and 5-LOX played an important role in chronic inflammation involving pain, destruction of bone and cartilage that can lead to severe disability. The levels of COX-2 and 5-LOX in PBMC were analyzed with ELISA immunoassay (Fig. 6). There was a
Fig. 3. Effects of EXS on arthritis as assessed with arthritic scores in rats. Paws swelling were evaluated and graded as arthritis scores for severity and loci of erythema, swelling and induration. The normal group rats was 0 score and other groups were evaluated using a 5-point scale: 0 ¼ no signs of disease, 1¼ signs involving the ankle/wrist, 2¼ signs involving the ankle plus tarsal of the hind paw and/or wrist plus carpals of the forepaw, 3 ¼ signs extending to the metatarsals or metacarpals, and 4 ¼severe disease involving the entire hind or fore paw. The arthritis score record every three days. The maximum arthritic score per rat was set at 8 (4 points 2 hind paws). The data represent the mean 7 S.D. (n¼ 8). Values are statistically significant at np o 0.05, nnp o0. 01 compared with the control group.
Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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significant elevation of COX-2 and 5-LOX levels of control group rats compared with the normal group rats. However, the level of COX-2 and 5-LOX were reduced after treatment with EXS (300, 75 mg/kg) and MTX. 3.5. Effects of EXS on relative cytokine production in the serum of AA rats
Fig. 4. Relationship between the extent of joint inflammation and the degree of weight loss. The weight of all rats was observed once a week, the weight before CFA-immunized recorded as initial weight. The weight increment calculated as follows: Δ (g) ¼ weight (day 7, 14, 21, 28) – initial weight (day 0). The normal group rats showed normal weight growth and the control group rats showed weight growth affect with CFA-induce arthritis. Those MTX or EXS (300, 75 mg/kg) treatment compared with normal group. The data represent the mean 7 S.D. (n¼ 8). Values are statistically significant at np o0.05, nnp o 0. 01 compared with the control group.
Cytokines such as IL-1β, TNF-α, IL-6 and IL-10 play an important role the pathogenesis of rheumatoid arthritis. Therefore, levels of these cytokines in the serum of AA rats were analyzed by ELISA kits (Fig. 7). As shown in the results, the levels of proinflammatory cytokine TNF-α, IL-1β and IL-6 in control group rats were significantly elevated while anti-inflammatory cytokine IL-10 was declined compared with the normal group rats. Decreasing in levels of TNF-α and IL-1β and increasing in IL-10 in serum of rats were observed in EXS treatment group (300, 75 mg/kg) (po0.05). The same effect was observed in MTX (3 mg/kg) treatment group. However, both concentrations of EXS treatment groups did not show any significant reduction in the IL-6 level when compared with the control group. 3.6. Effect of EXS on histopathological changes RA is well characterized with synovial hyperplasia, pannus formation, cartilage and bone destruction in the joint. In our
Fig. 5. Effects of EXS on thymus index and spleen index of AA rats. The thymus and spleen were promptly removed and weighed when the animals sacrificed at day 28 after immunization. The thymus index and spleen index were expressed as the ratio (mg/g) which calculated as follows: ratio (mg/g) ¼[thymus or spleen weight (mg)/body weight (g)] n103. The data represent the mean 7 S.D. (n¼ 8). Values are statistically significant at np o 0.05, nnp o0. 01 compared with the control group.
Fig. 6. Effects of EXS on COX-2 and 5-LOX activity in rats PBMC. The peripheral blood mononuclear cells (PBMC) were isolated from the blood, which collected from rats when experiment finished on day 28. The COX-2 and 5-LOX levels in PBMC were determined using an ELISA immunoassay kit according to the manufacturer's instructions. The data represent the mean7 S.D. (n ¼8). Values are statistically significant at np o 0.05, nnp o 0. 01 compared with the control group.
Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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Fig. 7. Effects of EXS on cytokine production in serum. On day 28, the rats were sacrificed and the blood was collected when the observation finished. When the blood was standing for 30 min, the serum was collected by centrifugation at 3000 rpm for 10 min. The levels of TNF-α, IL-1β, IL-6 and IL-10 in serum were determined using an ELISA immunoassay kits according to the manufacturer's instructions. The data represent the mean7 S.D. (n¼ 8). Values are statistically significant at np o 0.05, nnp o 0. 01 compared with the control group.
histopathological evaluation by H&E staining, the knee joints of the model group rats displayed notable synovial hyperplasia, inflammatory cell infiltration into the joint capacity, and partial bone destruction (Fig. 8B). The rats treated with 300 and 75 mg/kg of EXS showed a remarkable reduction in synovial hyperplasia, inflammatory cell infiltration compared with control rats (Fig. 8D and E). The MTX treatment group showed a better effect in ameliorate of synovial hyperplasia, inflammatory cell infiltration compared with the EXS treatment group (Fig. 8C).
4. Discussion Herbal medicines have been widely used in oriental clinics to treat different diseases, such as RA, for thousands of years. In recent years, herbal medicines or their formula, which are important parts of complementary and alternative medicine (CAM) for RA, is widely used for preclinical or clinical study (Liu et al., 2013; Rao et al., 1999; Shin et al., 2003). Thus, herbal medicines constitute a potentially important avenue leading to novel therapeutic agents for RA that may not only prevent structural damage of arthritic joints caused by tissue and bone breakdown, but also be relatively safe, inexpensive, highly tolerated, and convenient for many patients (Liu et al., 2011). CFA-induced arthritis in experimental rats is a chronic inflammatory disease characterized by infiltration of the synovial membrane and associated with destruction of the joints resembling
closely to the human rheumatoid arthritis (Jasin et al., 1973), and it is the most widely used chronic test model in which the clinical and pathological changes are comparable with those seen in human rheumatoid arthritis (Shinde et al., 1999). Paw swelling and arthritic scores are index of measuring the anti-arthritic activity of various drugs and employed here to determine the activity of EXS at the dose level of 300 and 75 mg/kg/i.g. EXS administered groups showed marked reduction in paw volume and a decrease in arthritic scores when compared with the control group. Yoshikawa et al. (1985) found that there was significant weight loss from the day following the injection of the adjuvant, but thereafter normal weight gain in the rats was observed. The result of the present study also indicates that there is a close relationship between the extent of inflammation and loss of body weight. After administration of EXS, the treated rats exhibited a significant weight loss and inhibition of index of thymus. Chronic inflammation involves the release of a number of mediators which are responsible for the pain, destruction of bone and cartilage that can lead to severe disability (Eric and Lawrence, 1996). Prostaglandins are formed by the interaction of two distinct but related enzymes, COX-1 and COX-2. The constitutive enzyme COX-1 is responsible for maintaining normal renal function, gastric mucosa integrity and homeostasis and the inducible isoform. While COX-2, is unregulated in inflamed tissue and is therefore thought to be responsible for the enhanced production of prostaglandins (Griswold and Adams, 1996). The activity of COX-2 was decreased significantly in EXS treated groups at the dose of 300
Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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Fig. 8. Effect of EXS on histopathological changes. The rats were sacrificed via anesthesia after serum collected on day 28. Knee joints and hind paws were removed from the rats and fixed in 10% phosphate buffered formalin, decalcified in 10% EDTA for 30 days at 4 1C, then embedded in paraffin. Serial paraffin sections (5 mm) were stained with hematoxylin and eosin (H&E). (A) Normal group rats showed the normal articular cartilage, absence of damage in the synovium; (B) control group rats showed marked infiltration of inflammatory cells and synovial hyperplasia; (C) MTX (3 mg/kg) treatment group; (D) EXS 300 mg/kg treatment group; and (E) EXS 75 mg/kg treatment group. (C)–(G) showed less inflammatory cell infiltration, well preserved joint spaces and minimal synovia hyperplasia.
and 75 mg/kg. 5-LOX is the key enzyme involved in the synthesis of leukotriene from arachidonic acid. The decreased 5-LOX activities observed by the administration of EXS (300, 75 mg/kg) suggest that inhibition of leukotriene synthesis may be another mechanism through which EXS mediates its anti-inflammatory effect. The present study examined the inhibitory effects on adjuvant arthritis rats and its possible anti-inflammatory and immunomodulatory mechanisms of EXS. In particular, some immunemodulatory cytokines, such as TNF-α, IL-1β, IL-6 and IL-10, are documented as being critically important in RA in humans (Jacques et al., 2006; Sweeney and Firestein, 2004). The network of the cytokines, including pro-inflammatory and anti-inflammatory mediators, normally present in the steady state, is interrupted by various factors such as infectious agent and other environmental exposure in RA (Firestein, 2005; Sweeney and Firestein, 2004). Thus the large amount of pro-inflammatory cytokines is excreted by inflammatory cells accompanied by some anti-inflammatory mediators released to alleviate the inflammation. These cytokines play a crucial role both in the initiation and the perpetuation of the localized and systemic inflammatory changes (Hackett et al., 2008; Miossec, 2004). They contribute to many features of arthritic inflammation, including synovial tissue inflammation, synovial proliferation, and cartilage and bone damage. Among the proinflammatory factors, TNF-α, IL-1β and IL-6 are considered to be of great importance in the pathogenesis of RA, especially TNF-α. This seems to be on top of a cytokine cascade, and can increase the levels of IL-1β, GM-CSF and stimulate cartilage matrix degradation, while up-regulating the production of matrix degrading enzymes such as matrix metalloproteinase (MMPs) (Goldring, 2000; Smolen and Steiner, 2003). IL-1β and IL-6 have also been proposed to contribute to the development of arthritis, therefore, it has been suggested that inhibition of these cytokines might be an effective strategy for the treatment of RA. IL-10 has been
regarded as upstream regulators that control the progression of RA negatively (Apparailly et al., 1998; Joosten et al., 1999). IL-10 can not only inhibit cytokines produced by Th1 cells, such as IFN-γ, TNF-α, IL-1β and GM-CSF, but it can also inhibit IL-18 mRNA expression (Fiorentino et al., 1991). In our experiments, both doses of EXS significantly reduced the serum TNF-α and IL-1β levels. The level of IL-10 was markedly increased at doses of 300 and 75 mg/kg of the EXS treatment group. The results indicate that antiinflammatory effect of Xanthium strumarium could be associated with its inhibition TNF-α, IL-1β level and increasing IL-10 level. As reported, more than 70 compounds that include volatile oils, sesquiterpene lactones, glycoside, phenols, polysterols, thiazinediones and alkaloids have been isolated from the leaves and seeds of Xanthium strumarium (Kamboj and Saluja, 2010). In order to find the chemical responsible of Xanthium strumarium for its significant anti-inflammatory effect, we investigated the chemical profile of EXS with HPLC-DAD. Ten phenolic acid derivatives were identified from 14 detected peaks. As shown in the result, phenolic compounds were the main constituents in EXS, especially chlorogenic acid and 1,5-O-dicaffeoylquinic acid (Han et al., 2009). A few studies on the in vivo and in vitro anti-inflammatory and antinociceptive effects of Xanthium strumarium have been reported previously. A study of methanol extract of Xanthium strumarium (MXS) on lipopolysaccharide-induced NO, PGE2 and TNF-α production in RAW264.7 showed that the MXS inhibited the NO, PGE2 and TNF-α production and the iNOS and COX-2 level by inhibiting NF-κB DNA binding and translocation of NF-κB to the nucleus via blocking the degradation of inhibitor of κB-α. From the results of carrageenin-induced acute paw edema, acetic acid-induced abdominal constriction and hot plate test in mice, MXS also showed anti-inflammatory and anti-nociceptive activities in vivo (Kim et al., 2005). In another bioactivity-guided fractionation of anti-inflammatory and analgesic study, the result showed that the n-butanol fraction, which is rich in phenolic components, showed
Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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the highest anti-inflammatory activity in the croton-oil-induced ear edema test and anti-nociceptive effects in the acetic acid induced writhings test in mice (Han et al., 2007). Chlorogenic acid, which has been reported to possess anti-inflammatory activity (Dos Santos et al., 2006), was found high content in Xanthium strumarium in our lab (Han et al., 2009). However, in our extra study of anti-rheumatic effect of chlorogenic acid (data unpublished), it did not induce significant inhibition of the paw swelling, arthritis scores and cytokines, but decreased the inflammatory mediators (COX-2 and 5-LOX) in AA rats. It indicated that CGA showed potential anti-inflammatory activity, which was in accordance with the literature (Dos Santos et al., 2006), but this activity can no't contribute greatly to the anti-rheumatic effect of EXS. The paradigm of traditional Chinese herbal medicine (TCHM) emphasizes the importance of multi-compound, multi-ingredient preparations as being responsible for the activity of the herbal drug, in contrast to modern pharmacology and drug development that often focus on a single chemical entity (Xie et al., 2006). Thus, a single compound isolated from an herbal medicine cannot reflect the effect of the whole plant, especially in the treatment of a chronic progressive disease with systemic symptoms. In summary, the results of the present study suggest that ethanol extract of Xanthium strumarium is effective on CFA induced arthritis in rats. The anti-arthritic activity of extract of Xanthium strumarium is probably related to: (1) Decrease in the spleen index; (2) downregulation in the levels of COX-2, 5-LOX and pro-inflammatory cytokines TNF-α, IL-1β in the serum of rats with CFA, and (3) up-regulation in the concentration of the anti-inflammatory cytokine IL-10 in serum. Xanthium strumarium can be regarded as a potential candidate for use in general therapeutics and as an immunemodulatory medicine in RA. However, the active constituent group and the synergy effect of multi-component need to be investigated further.
Acknowledgments This paper was supported by TCM Modernization Foundation of Science and Technology Commission of Shanghai (Nos. 10DZ1972000; 13401900102), National Natural Science Foundation of China (No. 81102773). Reference Apparailly, F., Verwaerde, C., Jacquet, C., Auriault, C., Sany, J., Jorgensen, C., 1998. Adenovirus-mediated transfer of viral IL-10 gene inhibits murine collageninduced arthritis. Journal of Immunology 160, 5213–5220. Chopra, R., Nayar, S., C.I., 1986. Glossary of Indian medicinal plants. Council of Scientific and Industrial Research, 259. Cumanda, J., Marinoni, G., De Bernardi, M., Vidari, G., Finzi, P.V., 1991. New Sesquiterpenes from Xanthium catharticum. Journal of Natural Products 54, 460–465. Dos Santos, M.D., Almeida, M.C., Lopes, N.P., et al., 2006. Evaluation of the antiinflammatory, analgesic and antipyretic activities of the natural polyphenol chlorogenic acid[J]. Biological and Pharmaceutical Bulletin 29 (11), 2236. Eric, G.B., Lawrence, J.L., 1996. Rheumatoid Arthritis and Its Therapy, the Textbook of Therapeutic Drugs and Disease Management. Williams and Wilkins Company, Baltimore. Fiorentino, D.F., Zlotnik, A., Vieira, P., Mosmann, T.R., Howard, M., Moore, K.W., O'Garra, A., 1991. IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. Journal of immunology 146, 3444–3451. Firestein, G.S., 2005. Immunologic mechanisms in the pathogenesis of rheumatoid arthritis. Journal of Clinical Rheumatology: Practical Reports on Rheumatic and Musculoskeletal Diseases 11, S39–S44. Goldring, M.B., 2000. The role of the chondrocyte in osteoarthritis. Arthritis and Rheumatism 43, 1916–1926. Griswold, D.E., Adams, J.L., 1996. Constitutive cyclooxygenase (COX-1) and inducible cyclooxygenase (COX-2): rationale for selective inhibition and progress to date. Medicinal Research Reviews 16, 181–206.
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Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats Bing Lin a,1, Ping Han b,1, Wei Yue a, Xue-Qing Ma c, Khalid Rahman d, Cheng-Jian Zheng a, Lu-Ping Qin a,n, Ting Han a,nn a
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, PR China Center for Disease Control and Prevention, Jinan Military Region, PLA, Ji'nan 250014, PR China c Department of Pharmaceutical analysis, School of Pharmacy, NingXia Medical University, 1160 Shenli Street, Yinchuan 750004, PR China d Faculty of Science, School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 July 2013 Received in revised form 7 May 2014 Accepted 18 May 2014
Ethnopharmacological relevance: Xanthium strumarium L. fruit (Xanthiu fruit) has been traditionally used as a medicinal herb in China for the treatment of many ailments including rheumatoid arthritis. However, the anti-arthritic activity of Xanthium strumarium fruit has still not been demonstrated. In the present study, we confirmed that the extract of Xanthium strumarium (EXS) prevents rheumatoid arthritis induced by Complete Freund's Adjuvant (CFA) in rats. Materials and methods: Male Wistar rats (1607 10 g) were immunized by intradermal injection of 0.1 mL of CFA into the left hind metatarsal footpad. EXS was administered orally at a dose of 300 and 75 mg/kg once a day after the induction of adjuvant arthritis. Methotrexate (3 mg/kg, twice a week) was used as a positive control. Paw swelling, arthritic score, body weight loss, spleen index, thymus index, serum cytokines, inflammatory mediators and histological change were measured. The chemical profile of EXS was analyzed by HPLC-DAD. Results: We found that the EXS significantly suppressed paw swelling and arthritic score, increased body weight loss and decreased the thymus index The overproduction of TNF-α and IL-1β was remarkably suppressed in the serum of all EXS-treated rats, and in contrast IL-10 was markedly increased. The level of COX-2 and 5-LOX was also decreased with EXS treatment. Ten phenolic acid derivatives were identified from 14 detected peaks by HPLC-DAD with the reference substances and verified by LC–MS. Conclusions: These results suggest the potential effect of EXS as an anti-arthritis agent towards CFAinduced arthritis in rats. Xanthium strumarium has the potential to be regarded as a candidate for use in general therapeutics and as an immune-modulatory medicine in rheumatoid arthritis. & 2014 Published by Elsevier Ireland Ltd.
Keywords: Xanthium strumarium L. Adjuvant arthritis Inflammation Anti-arthritis
1. Introduction Rheumatoid arthritis (RA) is a chronic destructive disease of the joints and is characterized by chronic proliferative synovitis, infiltration of inflammatory cells into the synovial tissue of joints, and cartilage destruction. It can rapidly progress into a multisystem inflammation with irreversible joint destruction, thus increasing the risk of mortality (Harris, 1990; Praveen and Suchita, 2013). Over 60 million adults in the world wide are affected by this disease and its prevalence is three times more in
n
Corresponding author. Tel./fax: þ 86 21 81871300. Corresponding author. Tel./fax: þ86 21 81871306. E-mail addresses: [email protected] (L.-P. Qin), [email protected] (T. Han). 1 Bing Lin and Ping Han equally contributed to this manuscript; both should be considered as first author. nn
women than men. The age of onset is between late twenties and early fifties, however, no age is immune to the disease (Winters and Geissman, 1969). The life span of patients with RA is shortened due to an increase in cardiovascular and cerebrovascular diseases (Wolfe et al., 1994). The non-steroidal anti-inflammatory drugs (NSAIDs) and biologics (e.g., TNF-α antibody and the decoy TNF-α receptor) represent a prominent group of drugs used in the treatment of RA. However, the usage of these drugs is associated with severe adverse effects, including gastrointestinal bleeding and cardiovascular complications (Winters and Geissman, 1969). Therefore, more and more patients are experimenting with medical botany options in order to cope with this debilitating disease. A recent investigation has estimated that 60–90% of patients with rheumatoid arthritis are very likely to use botanicals (Rao et al., 1999). The genus Xanthium (Family Asteraceae) is represented by 25 species in the world, of which three species and one variety
http://dx.doi.org/10.1016/j.jep.2014.05.023 0378-8741/& 2014 Published by Elsevier Ireland Ltd.
Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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are found abundantly throughout China (Han et al., 2007). Historically, Xanthium strumarium L., an important medicinal plant in the Xanthium species, has been used as reputed herbal medicines in China, Europe, Indo-China, Malaysia and America (Kamboj and Saluja, 2010). The whole plant is used as a medicine for curing nasal sinusitis, headache, urticaria and arthritis. It has also been reported to possess curative effects against chronic bronchitis, chronic rhinitis, allergic rhinitis, lumbago and other ailments (Zhu, 1998) and is used by various native American tribes to relieve constipation, diarrhea and vomiting (Kamboj and Saluja, 2010). An infusion of the plant has also been used in the treatment of rheumatism, diseased kidneys and tuberculosis (Chopra and Nayar, 1986). Additionally, Xanthium strumarium tissues contain high amounts of bioactive compounds such as glycosides, phytosterols, phenolic acids and xanthiazones, and the extract of this herb exhibit analgesic, antibacterial, antifungal, antimalarial, antirheumatic, antispasmodic, antitussive, cytotoxic, hypoglycemic and stomachic properties (Cumanda et al., 1991; Mahmoud, 1998; Qin et al., 2006; Winters and Geissman, 1969). In our previous studies of croton-oil-induced ear edema test and acetic acidinduced writhing test, it was shown that oral administration of EXS to mouse can attenuate inflammatory ear edema as well as reducing the number of writhing induced by acetic acid in mice in a dose-dependent manner (Han et al., 2007). The traditional use of Xanthium strumarium as an anti-inflammatory agent may have potential anti-arthritic effects in the treatment of RA, although as far as we know, no studies have focused on the in vivo effects of this herb. Therefore, the current study was designed to confirm the anti-arthritic effect and explore the potential mechanism of Xanthium strumarium on adjuvant-induced arthritis (AA) in rats, an animal model of rheumatoid arthritis.
water with 1% w/v sodium carboxyl methylcellulose (Na-CMC). The doses employed are expressed as mg of dried extract per kg body weight. 2.4. HPLC-DAD analysis of EXS An Agilent 1100 series HPLC-DAD system comprising a vacuum degasser, binary pump, autosampler, thermostated column compartment and DAD (Agilent, USA) was used for acquiring chromatograms and UV spectra. A Zorbax SB C-18 reversed-phase column (250 mm 4.6 mm, 5 μm) together with a Phenomenex C-18 guard column (10 mm 4.6 mm, 5 μm) were used with column temperature set at 25 1C. The mobile phase consisted of 0.5% (v/v) phosphoric acid in water (A) and methanol (B). Separation was achieved by a linear gradient elution from 20–40% B in 0–20 min, 40–50% B in 20–50 min and 50% B in 50–60 min at a flow rate of 0.8 mL/min. The DAD detector wavelength was set at 327 nm for acquiring chromatograms. Ultra-violet (UV) spectra were acquired from 200 to 700 nm. EXS prepared as mentioned above. Ten reference substances were used for the qualitative analysis: 1,3,5-O-tricaffeoylquinic acid, 1-O-caffeoylquinic acid, 1,4-Odicaffeoylquinic acid, 3-O-caffeoylquinic acid, chlorogenic acid, 4-O-caffeoylquinic acid, 1,3-O-dicaffeoylquinic acid and 1,4-Odicaffeoylquinic acid were isolated and purified from the fruits of Xanthium strumarium in our laboratory; 1,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid and 3,4,5-O-tricaffeoylquinic acid were purchased from National Institute for the Control of Drug and Biological Products (Beijing, China). The compounds were identified or deducted based on comparing individual peak retention times with the reference substances and verified by LC–MS (Han et al., 2009). 2.5. Animals
2. Materials and methods 2.1. Reagents and apparatus Agilent 1100 Series High Performance Liquid ChromatographyDiode Array Detector (HPLC-DAD) (Agilent, USA), Plethysmometer 7140 (UGO BASILE, Italy), FLx800 Fluorescence Microplate Reader (Bio-Tek, USA); Histopaque 1083 and Complete Freund's adjuvant (CFA) were purchased from Sigma (Sigma Chemical Co., USA), Methotrexate (Shanghai Sine Pharmaceutical Co., Ltd., China). IL-1β, TNF-α, IL-6 and IL-10 ELISA kits were purchased from the R&D system (R&D system, USA), COX-2 and 5-LOX ELISA kits were obtained from Biocalvin (Biocalvin, Suzhou, China). All the other chemicals and biochemical used were of the highest grade available.
Male Wistar rats, aged 6–8 weeks (160 710 g), were purchased from the Experimental Animal Center of Second Military Medical University (Shanghai, China). All rats were allowed to acclimatize for 1 week before the experiments were started. The rats were housed under standard laboratory conditions (room temperature 2572 1C, relative humidity 40–70% and free access to water) maintained on a 12-h light 12-h dark cycle. This experiment was approved by the Bioethics Committee of the Second Military Medical University (Shanghai, China), and the procedures of the experiment strictly adhered to generally accepted international rules and regulations. 2.6. Induction of adjuvant arthritis and treatment
The ripe fruits of Xanthium strumarium, Xanthii Fructus were purchased from an Herbal Medicinal Materials Company of Anton pharmaceutical Co., Ltd. (Bozhou, Anhui province, China), and authenticated by Prof. H.C. Zheng (The Second Military Medical University, Republic of China). A voucher specimen has been deposited in School of Pharmacy, Second Military Medical University, Shanghai, China.
Complete Freund's Adjuvant (CFA, sigma) was prepared by suspending heat-killed BCG in liquid paraffin at 10 mg/mL. The Wistar rats were immunized by intradermal injection of 0.1 mL of CFA into the left hind metatarsal footpad of rat (Perera et al., 2011). The rats were divided into 5 groups randomly, in which the rats with AA received EXS at 300 and 75 mg/kg once daily by intragastric (i.g.) from day 2. Methotrexate (3 mg/kg, twice a week) was used as a positive control. The normal and control group rats were given an equal volume of vehicle (1% CMC-Na) at the same time.
2.3. Preparation of plant extract
2.7. Assessment of arthritis
Dried fruits of Xanthii Fructus (5 kg) were extracted with 75% ethanol at 70 1C for 3 times (2 h each time). The extract was then combined and evaporated to dryness under the reduced pressure (60 1C), and yielded 300 g residue (Extract of Xanthium strumarium fruits, EXS). For pharmacological studies, EXS was suspended in
The volume of the hind paw swelling was measured with electronic water plethysmographicall before first immunization (basic value, day 0) and repeat on days 7, 14, 21 and 28. Meanwhile the body weight of rats was measured every 7 days, and the changes in body weight are shown as weight growth (g). Arthritis
2.2. Plant material
Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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score was used to evaluate the clinical severity of AA rats. A wellestablished, widely used scoring system was used to evaluate the severity of AA (Zhang et al., 2009). Paws were examined and graded for severity and loci of erythema, swelling and induration using a 5-point scale: 0 ¼no signs of disease, 1¼ signs involving the ankle/wrist, 2¼ signs involving the ankle plus tarsal of the hind paw and/or wrist plus carpals of the forepaw, 3 ¼signs extending to the metatarsals or metacarpals, and 4 ¼severe disease involving the entire hind or fore paw. The maximum arthritic score per rat was set at 8 (4 points 2 hind paws).
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Diluent were added into the wells of a microtiter plate coated with rat COX-2 antibody, and then 100 μL HRP-conjugate reagent was added and incubated for 1 h at 37 1C. After washing with Wash Solution (400 μL) for 4 times, chromogen solution A and B each 50 μL were added and incubated for 15 min at 37 1C, protected from light. Then, added Stop Solution 50 μL and the optical density were calculated with an automated Colter microplate reader at 450 nm. The same procedure was used to assay 5-LOX, with the substitution of antibodies specific for this enzyme. 2.11. Measurement of serum cytokines concentrations
2.8. Thymus index and spleen index assay The thymus and spleen were promptly removed and weighed when the animals sacrificed at day 28 after immunization (Pentobarbital Sodium, 40 mg/kg; i.p.). The thymus index and spleen index were expressed as the ratio (mg/g) of thymus and spleen wet weight versus body weight, respectively (Zhang et al., 2004). 2.9. Isolation of peripheral blood mononuclear cells (PBMC) The method of isolation of peripheral blood mononuclear cells was modified from Radhika et al. (2007). In brief, Histopaque 1083 solutions (3 mL) were placed in a 15 mL tube and 3 mL blood was layered on top of this density gradient. The blood cells were separated into two fractions after centrifugation 1500 rpm for 30 min: the mononuclear cells were in the upper white layer plus the majority of platelets at the interface region, and the erythrocytes and granulocytes were in the lower layer. The plasma layer on top with no cells was removed and discarded. The monocytes from the buffy coat were carefully taken off by aspiration and washed with phosphate buffered saline (PBS). The pellet was resuspended in PBS-Tween and subjected to freeze-thaw cycle three times and the resulting lysate was used as the enzyme source. 2.10. Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) level in PBMC
When the blood was standing for 30 min, the serum was collected by centrifugation at 3000 rpm for 10 min and stored at 20 1C prior to analysis. The concentrations of cytokines such as TNF-α, IL-1β, IL-6 and IL-10 were quantified by radioimmunoassay according to the manufacturer's protocol. All samples assayed for TNF-α were measured using the test kit individually. The same procedure was used to assay IL-6, IL-1β and IL-10, with the substitution of antibodies specific to the cytokines. 2.12. Histological changes When the rats were sacrificed via anesthesia after serum collected on day 28. Knee joints and hind paws were removed from the rats for histological analysis. The joints were fixed in 10% phosphate buffered formalin, decalcified in 10% EDTA for 30 days at 4 1C, and then embedded in paraffin. Serial paraffin sections (5 mm) were stained with hematoxylin and eosin (H&E). 2.13. Statistical analysis The data represent the mean 7S.D. (n ¼ 8). Differences between experimental groups were tested using one-way ANOVA; p values less than 0.05 were considered significant.
3. Results 3.1. HPLC-DAD qualitative analysis
The COX-2 expression level in PBMC was determined using an ELISA immunoassay kit according to the manufacturer's instructions (Biocalvin). In brief, the PBMC lysate 10 μL and 40 μL Sample
The HPLC-DAD analysis of EXS (Fig. 1) shown that over 14 peaks were detected within 60 min, among which 10 compounds were
Fig. 1. HPLC-DAD analysis of EXS. HPLC-DAD was used for the qualitative analysis of the main constituents of the EXS. A total of 14 peaks were detected in the EXS, and 10 compounds were deduced based on comparing individual peak retention times with those of the authentic reference substances and verified by LC–MS. There were (1) 1-Ocaffeoylquinic acid; (2) unknown; (3) 3-O-caffeoylquinic acid; (4) chlorogenic acid; (5) 4-O-caffeoylquinic acid; (6) 1,3-O-dicaffeoylquinic; (7) unknown; (8) unknown; (9) 1,4-O-dicaffeoylquinic acid; (10) 1,5-O-dicaffeoylquinic acid; (11) 4,5-O-dicaffeoylquinic acid; (12) 1,3,5-O-tricaffeoylquinic acid; (13) 3,4,5-O-tricaffeoylquinic acid; and (14) unknown.
Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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identified or deducted based on comparing individual peak retention times with those of the authentic reference substances and verified by LC–MS. They were (1) 1-O-caffeoylquinic acid, (3) 3-Ocaffeoylquinic acid, (4) chlorogenic acid, (5) 4-O-caffeoylquinic acid, (6) 1,3-O-dicaffeoylquinic acid, (9) 1,4-O-dicaffeoylquinic acid, (10) 1,5-O-dicaffeoylquinic acid, (11) 4,5-O-dicaffeoylquinic acid, (12) 1,3,5-O-tricaffeoylquinic acid and (13) 3,4,5-Otricaffeoylquinic acid. 3.2. Effects of EXS on paw swelling, arthritis score and weight change Obvious joint swelling was shown in all immunized rats after 24 h. An initial development in the manifestations of inflammation from day 1 to day 4 was observed, and then it showed a brief decrease in the inflammatory signs from day 5 to day 6. This phase
was followed by a quick reappearance of inflammation on day 11 and day 12. Significant increasing of paw volume and arthritic scores were observed in CFA immunized rats when compared with the normal control rats. However, the polyarthritis symptoms and hind paw swelling reduced after treatment with EXS and MTX on day 14, 21 and 28. Overall, the reduced paw volume after treatment with both concentrations of EXS and MTX showed no significant differences from the normal group, while the level was significantly (po0.01) lower than the control group, the same results were observed on day 21 and day 28 (Fig. 2). All rats showed a significant increase of arthritic scores upon induction by CFA before day 10, however the rats treated with either EXS (both concentrations) or MTX successfully reduced the arthritic scores in rats compared with the control rats significantly from day 10 to day 28 (Fig. 3). The relationship between the extent of joint inflammation and the weight loss were investigated. As shown in Fig. 4, in the second week after adjuvant injection, the control group rats showed a significant weight loss (p o0.05), followed by normal weight gain in the subsequent weeks, whereas the EXS and MTX treatment groups did not show significant weight loss. 3.3. Effects of EXS on thymus index and spleen index
Fig. 2. Effects of EXS on paw swelling in rats. 40 male Wistar rats (160 7 10 g, 8/group) were divided into 5 groups randomly and immunized by intradermal injection of 0.1 mL of CFA into the left hind metatarsal footpad except normal group. The MTX group received methotrexate twice a week at 3 mg/kg as a positive control. The EXS group (300, 75 mg/kg) was received EXS once daily by intragastric (i.g.) from day 2. The volumes of the hind paw swelling (mL) were measured before first immunization (basic value, day 0) and repeat on days 7, 14, 21 and 28. The increment of paw swelling calculates as follows: Δ (mL) ¼volume day 14 (21 or 28) – volume day 0; Data represent the mean 7S.D. (n¼ 8). Values are statistically significant at np o 0.05, nnp o 0. 01 compared with the control group.
The thymus index and spleen index are associated with immunological functions. The thymus index and spleen index were assayed when the rats sacrificed at day 28, and the results showed that thymus index and spleen index of the control group were increased compared with normal group. Both concentrations of EXS (300, 75 mg/kg) showed a marked decreased of thymus index. However, there was no significant effect of spleen index in both concentrations of EXS treatment (Fig. 5). Both thymus index and spleen index were decreased in the MTX treatment group. 3.4. Effects of EXS on inflammatory mediators COX-2 and 5-LOX played an important role in chronic inflammation involving pain, destruction of bone and cartilage that can lead to severe disability. The levels of COX-2 and 5-LOX in PBMC were analyzed with ELISA immunoassay (Fig. 6). There was a
Fig. 3. Effects of EXS on arthritis as assessed with arthritic scores in rats. Paws swelling were evaluated and graded as arthritis scores for severity and loci of erythema, swelling and induration. The normal group rats was 0 score and other groups were evaluated using a 5-point scale: 0 ¼ no signs of disease, 1¼ signs involving the ankle/wrist, 2¼ signs involving the ankle plus tarsal of the hind paw and/or wrist plus carpals of the forepaw, 3 ¼ signs extending to the metatarsals or metacarpals, and 4 ¼severe disease involving the entire hind or fore paw. The arthritis score record every three days. The maximum arthritic score per rat was set at 8 (4 points 2 hind paws). The data represent the mean 7 S.D. (n¼ 8). Values are statistically significant at np o 0.05, nnp o0. 01 compared with the control group.
Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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significant elevation of COX-2 and 5-LOX levels of control group rats compared with the normal group rats. However, the level of COX-2 and 5-LOX were reduced after treatment with EXS (300, 75 mg/kg) and MTX. 3.5. Effects of EXS on relative cytokine production in the serum of AA rats
Fig. 4. Relationship between the extent of joint inflammation and the degree of weight loss. The weight of all rats was observed once a week, the weight before CFA-immunized recorded as initial weight. The weight increment calculated as follows: Δ (g) ¼ weight (day 7, 14, 21, 28) – initial weight (day 0). The normal group rats showed normal weight growth and the control group rats showed weight growth affect with CFA-induce arthritis. Those MTX or EXS (300, 75 mg/kg) treatment compared with normal group. The data represent the mean 7 S.D. (n¼ 8). Values are statistically significant at np o0.05, nnp o 0. 01 compared with the control group.
Cytokines such as IL-1β, TNF-α, IL-6 and IL-10 play an important role the pathogenesis of rheumatoid arthritis. Therefore, levels of these cytokines in the serum of AA rats were analyzed by ELISA kits (Fig. 7). As shown in the results, the levels of proinflammatory cytokine TNF-α, IL-1β and IL-6 in control group rats were significantly elevated while anti-inflammatory cytokine IL-10 was declined compared with the normal group rats. Decreasing in levels of TNF-α and IL-1β and increasing in IL-10 in serum of rats were observed in EXS treatment group (300, 75 mg/kg) (po0.05). The same effect was observed in MTX (3 mg/kg) treatment group. However, both concentrations of EXS treatment groups did not show any significant reduction in the IL-6 level when compared with the control group. 3.6. Effect of EXS on histopathological changes RA is well characterized with synovial hyperplasia, pannus formation, cartilage and bone destruction in the joint. In our
Fig. 5. Effects of EXS on thymus index and spleen index of AA rats. The thymus and spleen were promptly removed and weighed when the animals sacrificed at day 28 after immunization. The thymus index and spleen index were expressed as the ratio (mg/g) which calculated as follows: ratio (mg/g) ¼[thymus or spleen weight (mg)/body weight (g)] n103. The data represent the mean 7 S.D. (n¼ 8). Values are statistically significant at np o 0.05, nnp o0. 01 compared with the control group.
Fig. 6. Effects of EXS on COX-2 and 5-LOX activity in rats PBMC. The peripheral blood mononuclear cells (PBMC) were isolated from the blood, which collected from rats when experiment finished on day 28. The COX-2 and 5-LOX levels in PBMC were determined using an ELISA immunoassay kit according to the manufacturer's instructions. The data represent the mean7 S.D. (n ¼8). Values are statistically significant at np o 0.05, nnp o 0. 01 compared with the control group.
Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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Fig. 7. Effects of EXS on cytokine production in serum. On day 28, the rats were sacrificed and the blood was collected when the observation finished. When the blood was standing for 30 min, the serum was collected by centrifugation at 3000 rpm for 10 min. The levels of TNF-α, IL-1β, IL-6 and IL-10 in serum were determined using an ELISA immunoassay kits according to the manufacturer's instructions. The data represent the mean7 S.D. (n¼ 8). Values are statistically significant at np o 0.05, nnp o 0. 01 compared with the control group.
histopathological evaluation by H&E staining, the knee joints of the model group rats displayed notable synovial hyperplasia, inflammatory cell infiltration into the joint capacity, and partial bone destruction (Fig. 8B). The rats treated with 300 and 75 mg/kg of EXS showed a remarkable reduction in synovial hyperplasia, inflammatory cell infiltration compared with control rats (Fig. 8D and E). The MTX treatment group showed a better effect in ameliorate of synovial hyperplasia, inflammatory cell infiltration compared with the EXS treatment group (Fig. 8C).
4. Discussion Herbal medicines have been widely used in oriental clinics to treat different diseases, such as RA, for thousands of years. In recent years, herbal medicines or their formula, which are important parts of complementary and alternative medicine (CAM) for RA, is widely used for preclinical or clinical study (Liu et al., 2013; Rao et al., 1999; Shin et al., 2003). Thus, herbal medicines constitute a potentially important avenue leading to novel therapeutic agents for RA that may not only prevent structural damage of arthritic joints caused by tissue and bone breakdown, but also be relatively safe, inexpensive, highly tolerated, and convenient for many patients (Liu et al., 2011). CFA-induced arthritis in experimental rats is a chronic inflammatory disease characterized by infiltration of the synovial membrane and associated with destruction of the joints resembling
closely to the human rheumatoid arthritis (Jasin et al., 1973), and it is the most widely used chronic test model in which the clinical and pathological changes are comparable with those seen in human rheumatoid arthritis (Shinde et al., 1999). Paw swelling and arthritic scores are index of measuring the anti-arthritic activity of various drugs and employed here to determine the activity of EXS at the dose level of 300 and 75 mg/kg/i.g. EXS administered groups showed marked reduction in paw volume and a decrease in arthritic scores when compared with the control group. Yoshikawa et al. (1985) found that there was significant weight loss from the day following the injection of the adjuvant, but thereafter normal weight gain in the rats was observed. The result of the present study also indicates that there is a close relationship between the extent of inflammation and loss of body weight. After administration of EXS, the treated rats exhibited a significant weight loss and inhibition of index of thymus. Chronic inflammation involves the release of a number of mediators which are responsible for the pain, destruction of bone and cartilage that can lead to severe disability (Eric and Lawrence, 1996). Prostaglandins are formed by the interaction of two distinct but related enzymes, COX-1 and COX-2. The constitutive enzyme COX-1 is responsible for maintaining normal renal function, gastric mucosa integrity and homeostasis and the inducible isoform. While COX-2, is unregulated in inflamed tissue and is therefore thought to be responsible for the enhanced production of prostaglandins (Griswold and Adams, 1996). The activity of COX-2 was decreased significantly in EXS treated groups at the dose of 300
Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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Fig. 8. Effect of EXS on histopathological changes. The rats were sacrificed via anesthesia after serum collected on day 28. Knee joints and hind paws were removed from the rats and fixed in 10% phosphate buffered formalin, decalcified in 10% EDTA for 30 days at 4 1C, then embedded in paraffin. Serial paraffin sections (5 mm) were stained with hematoxylin and eosin (H&E). (A) Normal group rats showed the normal articular cartilage, absence of damage in the synovium; (B) control group rats showed marked infiltration of inflammatory cells and synovial hyperplasia; (C) MTX (3 mg/kg) treatment group; (D) EXS 300 mg/kg treatment group; and (E) EXS 75 mg/kg treatment group. (C)–(G) showed less inflammatory cell infiltration, well preserved joint spaces and minimal synovia hyperplasia.
and 75 mg/kg. 5-LOX is the key enzyme involved in the synthesis of leukotriene from arachidonic acid. The decreased 5-LOX activities observed by the administration of EXS (300, 75 mg/kg) suggest that inhibition of leukotriene synthesis may be another mechanism through which EXS mediates its anti-inflammatory effect. The present study examined the inhibitory effects on adjuvant arthritis rats and its possible anti-inflammatory and immunomodulatory mechanisms of EXS. In particular, some immunemodulatory cytokines, such as TNF-α, IL-1β, IL-6 and IL-10, are documented as being critically important in RA in humans (Jacques et al., 2006; Sweeney and Firestein, 2004). The network of the cytokines, including pro-inflammatory and anti-inflammatory mediators, normally present in the steady state, is interrupted by various factors such as infectious agent and other environmental exposure in RA (Firestein, 2005; Sweeney and Firestein, 2004). Thus the large amount of pro-inflammatory cytokines is excreted by inflammatory cells accompanied by some anti-inflammatory mediators released to alleviate the inflammation. These cytokines play a crucial role both in the initiation and the perpetuation of the localized and systemic inflammatory changes (Hackett et al., 2008; Miossec, 2004). They contribute to many features of arthritic inflammation, including synovial tissue inflammation, synovial proliferation, and cartilage and bone damage. Among the proinflammatory factors, TNF-α, IL-1β and IL-6 are considered to be of great importance in the pathogenesis of RA, especially TNF-α. This seems to be on top of a cytokine cascade, and can increase the levels of IL-1β, GM-CSF and stimulate cartilage matrix degradation, while up-regulating the production of matrix degrading enzymes such as matrix metalloproteinase (MMPs) (Goldring, 2000; Smolen and Steiner, 2003). IL-1β and IL-6 have also been proposed to contribute to the development of arthritis, therefore, it has been suggested that inhibition of these cytokines might be an effective strategy for the treatment of RA. IL-10 has been
regarded as upstream regulators that control the progression of RA negatively (Apparailly et al., 1998; Joosten et al., 1999). IL-10 can not only inhibit cytokines produced by Th1 cells, such as IFN-γ, TNF-α, IL-1β and GM-CSF, but it can also inhibit IL-18 mRNA expression (Fiorentino et al., 1991). In our experiments, both doses of EXS significantly reduced the serum TNF-α and IL-1β levels. The level of IL-10 was markedly increased at doses of 300 and 75 mg/kg of the EXS treatment group. The results indicate that antiinflammatory effect of Xanthium strumarium could be associated with its inhibition TNF-α, IL-1β level and increasing IL-10 level. As reported, more than 70 compounds that include volatile oils, sesquiterpene lactones, glycoside, phenols, polysterols, thiazinediones and alkaloids have been isolated from the leaves and seeds of Xanthium strumarium (Kamboj and Saluja, 2010). In order to find the chemical responsible of Xanthium strumarium for its significant anti-inflammatory effect, we investigated the chemical profile of EXS with HPLC-DAD. Ten phenolic acid derivatives were identified from 14 detected peaks. As shown in the result, phenolic compounds were the main constituents in EXS, especially chlorogenic acid and 1,5-O-dicaffeoylquinic acid (Han et al., 2009). A few studies on the in vivo and in vitro anti-inflammatory and antinociceptive effects of Xanthium strumarium have been reported previously. A study of methanol extract of Xanthium strumarium (MXS) on lipopolysaccharide-induced NO, PGE2 and TNF-α production in RAW264.7 showed that the MXS inhibited the NO, PGE2 and TNF-α production and the iNOS and COX-2 level by inhibiting NF-κB DNA binding and translocation of NF-κB to the nucleus via blocking the degradation of inhibitor of κB-α. From the results of carrageenin-induced acute paw edema, acetic acid-induced abdominal constriction and hot plate test in mice, MXS also showed anti-inflammatory and anti-nociceptive activities in vivo (Kim et al., 2005). In another bioactivity-guided fractionation of anti-inflammatory and analgesic study, the result showed that the n-butanol fraction, which is rich in phenolic components, showed
Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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the highest anti-inflammatory activity in the croton-oil-induced ear edema test and anti-nociceptive effects in the acetic acid induced writhings test in mice (Han et al., 2007). Chlorogenic acid, which has been reported to possess anti-inflammatory activity (Dos Santos et al., 2006), was found high content in Xanthium strumarium in our lab (Han et al., 2009). However, in our extra study of anti-rheumatic effect of chlorogenic acid (data unpublished), it did not induce significant inhibition of the paw swelling, arthritis scores and cytokines, but decreased the inflammatory mediators (COX-2 and 5-LOX) in AA rats. It indicated that CGA showed potential anti-inflammatory activity, which was in accordance with the literature (Dos Santos et al., 2006), but this activity can no't contribute greatly to the anti-rheumatic effect of EXS. The paradigm of traditional Chinese herbal medicine (TCHM) emphasizes the importance of multi-compound, multi-ingredient preparations as being responsible for the activity of the herbal drug, in contrast to modern pharmacology and drug development that often focus on a single chemical entity (Xie et al., 2006). Thus, a single compound isolated from an herbal medicine cannot reflect the effect of the whole plant, especially in the treatment of a chronic progressive disease with systemic symptoms. In summary, the results of the present study suggest that ethanol extract of Xanthium strumarium is effective on CFA induced arthritis in rats. The anti-arthritic activity of extract of Xanthium strumarium is probably related to: (1) Decrease in the spleen index; (2) downregulation in the levels of COX-2, 5-LOX and pro-inflammatory cytokines TNF-α, IL-1β in the serum of rats with CFA, and (3) up-regulation in the concentration of the anti-inflammatory cytokine IL-10 in serum. Xanthium strumarium can be regarded as a potential candidate for use in general therapeutics and as an immunemodulatory medicine in RA. However, the active constituent group and the synergy effect of multi-component need to be investigated further.
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Please cite this article as: Lin, B., et al., Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.023i
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