Journal of Ethnopharmacology 152 (2014) 522–531

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Xylopia aethiopica (Annonaceae) fruit extract suppresses Freund's adjuvant-induced arthritis in Sprague-Dawley rats David D. Obiri a,n, Newman Osafo a, Patrick G. Ayande b, Aaron O. Antwi a a Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana b Department of Nursing, School of Biological Sciences, University of Cape Coast, Cape Coast, Ghana

art ic l e i nf o

a b s t r a c t

Article history: Received 27 November 2013 Received in revised form 20 January 2014 Accepted 29 January 2014 Available online 6 February 2014

Ethnopharmacological relevance: Xylopia aethiopica is used in a decoction of the dried fruit to treat bronchitis, asthma, arthritis, rheumatism, headache, neuralgia and colic pain. The aim of the study is to evaluate the anti‐arthritic effects of a 70% aqueous ethanol extract of the fruit of Xylopia aethiopica in a chronic inflammatory model. Materials and methods: Adjuvant arthritis was induced in Sprague-Dawley rats by intraplantar injection of Complete Freund's Adjuvant into the right hind paw. Foot volume was measured by water displacement plethysmometry. The oedema component of inflammation was evaluated as the percentage change in paw swelling and the total oedema induced calculated as area under the time course curves. In addition to X-ray radiography, histopathology of ankle joints supported by haematological analysis was used to assess the anti-arthritic action of the extract of Xylopia aethiopica (XAE). Results: Xylopia aethiopica extract (100, 300 and 600 mg kg
1) modified the time course curve significantly reducing hind paw oedema in the ipsilateral paw at all dose levels when administered both prophylactically and therapeutically. In addition XAE significantly suppressed the systemic spread of the arthritis from the ipsilateral to the contralateral limbs. The radiological pictures of the joints particularly metatarsal, phalanges and the ankle joint space of rats in the XAE-treated group showed protective effect against adjuvant-induced arthritis while histopathology revealed significant reduction in mononuclear infiltration, pannus formation and bone erosion. The haematological analysis in the test animals revealed significant improvement relative to the CFA model group. Conclusion: Xylopia aethiopica XAE suppresses joint inflammation and destruction in arthritic rats & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Xylopia aethiopica Inflammation Adjuvant-induced Rheumatoid arthritis Chemical compounds studied in this article: Aspirin (Acetylsalicylic acid) (PubChem CID: 2244)

1. Introduction Xylopia aethiopica [Dunal] A. Rich the “African guinea pepper” as it is commonly called is a tropical evergreen tree with seeds that are aromatic and contain bitter principles (Burkhill, 1985) and belongs to the Annonaceae family. Among the folkloric uses Igwe et al. (2003) report that a decoction of the plant is used to treat dysentery, bronchitis, ulceration, rheumatism, headache, neuralgia and colic pain. Results from the earlier and recent studies on

Abbreviations: DNA, deoxyribonucleic acid; ERK, extracellular signal-regulated kinase; HGB, haemoglobin; HCT, haematocrit; IgG, immunoglobulin G; IL, interleukin; MAPK, mitogen activated protein kinase; NO, nitric oxide; NF-κB, nuclear Factor-κB; PG, prostaglandin; RBC, red blood cell; RA, rheumatoid arthritis; TNFα, tumour necrosis factor α; WBC, white blood cell; XAE, xylopia aethiopica extract n Corresponding author. Tel.: þ233 3220 60372;/24 4573543; fax: þ233 3220 6063684. E-mail address: [email protected] (D.D. Obiri). http://dx.doi.org/10.1016/j.jep.2014.01.035 0378-8741 & 2014 Elsevier Ireland Ltd. All rights reserved.

extracts of the plant have shown that Xylopia aethiopica possess antibacterial (Asekun and Adeniyi, 2004; Fleischer et al., 2008; Kuete, 2010), antifungal (Tatsadjieu et al., 2003) and anti-plasmodial (Boyom et al., 2003) activities. Interestingly, while some extracts of Xylopia aethiopica reportedly exhibit antioxidant activity (Karioti et al., 2004) and protect rats from the adverse effects of irradiation (Adaramoye et al., 2010, 2011) some other extracts as reported by Ju et al. (2004) and Choumessi et al. (2012) demonstrate cytotoxic effects on a wide range of cancer cell lines through triggering DNA damage and accumulation of cells in the G1 phase of the cell cycle, followed by apoptosis. The constituent diterpenes called kauranes, notably kaurenoic acid (ent-kaur-16-en-19-oic acid) and xylopic acid [15β-acetoxy-(
)-kaur-16-en-19-oic acid] as elucidated by Ekong and Ogan (1968) account for the observed biological activities. In addition, kaurenoic acid exhibits analgesic (Block et al., 1998) diuretic, vasorelaxant, anti-inflammatory and anti-pyretic effects in rodents (Somova et al., 2001; Sosa-Sequera et al., 2010).

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Xylopia aethiopica possesses activities suggestive of benefit in a chronic inflammatory disorder such as rheumatoid arthritis (RA). For example, the constituent kauranes impair inflammation signalling through inhibition of the NF-κB signalling pathway (Castrillo et al., 2001) as a mechanism of anti-inflammatory action. In addition very recent in vivo studies demonstrating potent analgesic effects in three animal pain models; chemical (acetic acid-induced abdominal writhing and formalin tests), thermal (tail-flick and Hargreaves thermal hyperalgesia tests) and mechanical (Randall-Selitto paw pressure test) as reported by Woode et al. (2012) lend support to the potential utility of Xylopia aethiopica and its major constituent, xylopic acid in RA since pain is a cardinal sign of inflammation. RA is a reported autoimmune disease characterised by chronic inflammation of multiple joints. The classical features of RA are associated with subsequent progressive, erosive destruction of articular bone and cartilage, mononuclear cell infiltration, pannus formation, and functional impairment. Rat adjuvant- and collageninduced arthritis experimentally are the most widely used animal models of inflammatory polyarthritis and they present with clinical and pathological features typical of RA in human (Wooley, 2008; Bolon et al., 2011). Hoffmann et al. (1997) indicate that as in human RA, the prognosis of rat adjuvant-induced arthritis is divided into three phases; the induction phase which has no evidence of synovitis, followed by early synovitis, and finally late synovitis with progressive joint destruction. Although the initiating cause(s) of RA is not well established, Kishimoto et al. (1992) and Feldmann et al. (1996) report that elevated levels of pro-inflammatory cytokines notably TNFα and the activation of NF-κB signalling pathway are thought to be essential to the disease pathophysiology and progression. Thus, the reduction of pain, inflammation, and joint damage is the focus of drug treatment of RA. The anti-rheumatic drugs in clinical use are associated with severe adverse reactions and potential toxic effects (Amoroso et al., 2003). For instance at doses that would result in maximum efficacy, long-term use of the disease modifying anti-rheumatic drugs for the treatment of RA cause serious toxicities. The non-steroidal antiinflammatory drugs (NSAIDs) as reported by Wolfe et al. (1999) have little potential for modification of the disease progression at the doses that are generally safe for prolonged use in humans due to their associated gastrointestinal side effects. However, Sanghi et al. (2006) report that even though the selective cyclooxygenase (COX)-2 inhibitors minimise this gastrointestinal associated risks they also present unwanted side effects on the kidney and cardiovascular system. The steroidal drugs (glucocorticoids) on the other hand when taken chronically in doses needed for RA leave the patients with the so-called Cushing syndrome (Dore, 2010). In sum these biologic agents may improve RA but in addition to the severe adverse and toxic effects expensive costs also further limit their clinical application (Katikireddi et al., 2010). Consequently, natural herbal therapies have widely attracted attention in recent years (Venkatesha et al., 2011; de Sousa et al., 2012). As part of our on-going research into medicinal plants, we have recently reported that the aqueous ethanol extract of the dried fruit of Xylopia aethiopica has anti-allergic and anti-inflammatory actions in mice (Obiri and Osafo, 2013). In the present study we evaluate the effect of the dried fruit extract of Xylopia aethiopica on ameliorating arthritis in an experimental animal model of RA.

2. Materials and methods 2.1. Materials 2.1.1. Preparation of plant extract Samples of the dried fruits of Xylopia aethiopica [Dunal] A. Rich were purchased from a commercial herb market in Kumasi in

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March, 2012. The identity was confirmed as the fruit of Xylopia aethiopica [Dunal] A. Rich by anatomical observation and direct comparison with the authentic specimens, stored in the Herbarium in the Department of Herbal Medicine, KNUST, Kumasi. A voucher specimen (No. FP/09/77) has been deposited in the same department. Dried fruit (2.7 kg) was ground using heavy duty blender (37BL85 (240CB6), WARING Commercial, USA) and extraction was done with 70% v/v ethanol (5 L) by maceration for 24 h. The ethanol filtrate was concentrated under reduced pressure at 45 1C by a vacuum rotary evaporator (R-210, BUCHI, Switzerland) and further dried in an oven (Gallenkamp OMT, SANYO, Japan) to yield a solid mass of weight 167 g. The dried extract, XAE was freshly emulsified with Tween-80 and prepared with normal saline before use. 2.2. Experimental animals Purpose bred male Sprague-Dawley rats (200–250 g) were purchased from Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana and kept in the Animal facility of the Department of Pharmacology, College of Health Sciences, KNUST, Kumasi, Ghana. All animals were humanely handled throughout the experimental period in accordance with internationally accepted principles for laboratory animal use and care (EEC Directive of 1986: 86/609 EEC). Additionally all animal experiments were approved by the Department of Pharmacology, KNUST Ethics Committee. Therefore, animals were acclimated upon arrival for a week before assigned randomly to their respective group. Rats (5/cage) were housed in a polypropylene cage in a temperature-controlled room (22.2 1C) on a 12-h lightdark cycle with free access to commercial pellet diet (GAFCO, Ghana) and water ad libitum. All animals were euthanised at the end of each experiment. Each animal was therefore used only once. 2.3. Chemicals and reagents Aspirin was purchased from Sigma-Aldrich (St Louis, USA). Paraffin oil was purchased from KAMA Pharmaceutical Industries (Ghana). 2.4. Microorganism Heat-killed Mycobacterium tuberculosis [strains C, DT and PN (mixed)] was obtained from the Ministry of Agriculture, Fisheries and Food, UK. 2.5. Methods 2.5.1. Adjuvant-induced arthritis in rats Adjuvant arthritis was induced as previously described by Pearson (1956). Briefly, right hind paw of rats were injected intraplantar with 100 ml of Complete Freund's Adjuvant (CFA) prepared as a suspension of 5 mg ml
1 of heat-killed Mycobacterium tuberculosis [strains C, DT and PN (mixed)] in paraffin oil while non-arthritic control group received only intraplantar injection of 100 ml sterile paraffin oil referred to as Incomplete Freund's Adjuvant (IFA). Foot volume measurements were taken with a plethysmometer (Ugo Basile, Comerio, Italy) for both the ipsilateral (injected ) and the contralateral (non-injected) hind paws before intraplantar injection of CFA/IFA (day 0) and on every other day up to 28 days (Binder and Walker, 1998). The oedema component of inflammation was quantified by measuring the difference in foot volume between day 0 and the various time points. Foot volumes were individually normalised as percentage of change from their values at day zero and then averaged for each

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treatment group. To determine the mean percent change in paw volume for each treatment, the following equation was used:   PVt
PV0  100 ð1Þ % change in paw volume ¼ PV0 where PV0 and PVt are respectively the paw volumes before and at time T of the induction of the arthritis. Total oedema induced was determined as area under the time course curves (AUC). To determine the percent inhibition of the total oedema for each treatment, the following equation was used:  % inhibition

of oedema ¼

 AUCðcontrolÞ
AUCðdrug treatmentÞ  100 AUCðcontrolÞ

ð2Þ

2.5.1.1. Drug treatment and effects. In the preventive (prophylactic) protocol, drug-vehicle, XAE (100, 300 and 600 mg kg
1), or aspirin 100 mg kg
1 was given orally 1 h before the induction of the oedema on day zero and daily for 28 days while in the curative (therapeutic) protocol, treatment commenced on day 14 post oedema induction and daily till day 28. All drugs were freshly prepared on each day of oral drug administration. Drug effects were evaluated by comparing the maximal and total oedema responses attained during 28 days in drug-treated groups with the corresponding values attained in saline-treated inflamed control groups. Three indices of arthritic damage were used: oedema monitored through arthritis scoring and radiology (Xray), haematological analysis and histology. Data was presented as drug effects on the time-course and the total oedema response of adjuvant-induced arthritis for the 28-day period. All experiments were carried out in a double-blind manner and for consistency the same trained observer did all measurements throughout the study. Disease progression was monitored from day 0 to day 28 after which rats were sacrificed. 2.5.1.2. Arthritis score. The hind paw volume was used as a parameter for scoring. The severity of arthritic score was determined on extent of erythema and oedema of a given tissue (Cai et al., 2007) from photographs of the affected hind limbs taken with a digital camera (FE-5050, OLYMPUS, Tokyo, Japan) on day 28. The extent of inflammation from the photographs taken was blindly scored on a scale of 0–4 (0 – uninjected paw with no swelling, 1 – slight swelling and/or erythema, 2 – low to moderate oedema, 3 – pronounced oedema with limited joint use, 4 – excess oedema with joint rigidity). 2.5.1.3. X-ray radiography. Rats were anaesthetised by intraperitoneal injection of 50 mg kg
1 pentobarbitone sodium on day 28. Radiographs were taken with X-ray apparatus (PHILIPS Diagnose X-ray) operated at a voltage of 55 kV against 3.2 mA s
1 with a tube-to-film distance of 110 cm for lateral projection. The severity of the joint and bone deformation was blindly scored according to the extent of osteoporosis, joint spaces, osteophytes and joint structure (Pohlers et al., 2007) on a scale of 0–4 (0 – uninjected control group with no degenerative joint changes, 1 – slight soft tissue volume, joint space, subchondral erosion, periostitis, osteolysis, subluxation, and degenerative joint changes, 2 – low to moderate soft tissue volume, joint space, subchondral erosion, periostitis, osteolysis, subluxation, and degenerative joint changes, 3 – pronounced soft tissue volume, joint space, subchondral erosion, periostitis, osteolysis, subluxation, and degenerative joint changes, 4 – excess soft tissue volume, joint space, subchondral erosion, periostitis, osteolysis, subluxation, and degenerative joint changes).

2.5.1.4. Histopathology. The bones of the hind paws were excised on day 29 for histological analysis. Excised bones of the ipsilateral limbs were fixed in sterile Bouin's fluid (1% picric acid (88–89–1), 9.5% formaldehyde (50–00–0), 5% acetic acid (64–19–7), water) decalcified, sectioned and stained with haematoxylin and eosin to examine the histopathological changes during the experimental period under light microscope. The histopathological change of joints was blindly graded by a pathologist and assigned a score of 0–3 (0 – absence of synovial hyperplasia, pannus, bone erosion, fibrosis or presence of inflammatory cells as observed as neutrophils, 1 – minimal presence of synovial hyperplasia, pannus, bone erosion, fibrosis or presence of inflammatory cells as observed as neutrophils, 2 – mild/moderate presence of synovial hyperplasia, pannus, bone erosion, fibrosis or presence of inflammatory cells as observed as neutrophils, 3 – more intense presence of synovial hyperplasia, pannus, bone erosion, fibrosis or presence of inflammatory cells as observed as neutrophils). 2.5.1.5. Haematological analyses. Rats were euthanized and blood samples were collected from the jugular vein on day 29. A full blood count was done on the collected blood samples using blood analyser (Mindray BC 2800). The Biernacki reaction or eosinophil sedimentation rate (ESR) was also done as per the standard Westergren method using Westergren pipettes that meet the National Committee for Clinical Laboratory Standards' specification (NCCLS, 1993). 2.6. Statistical analysis All data are presented as the mean 7s.e.m. (n ¼5). The timecourse curves for paw volume were subjected to Two-way (treatment  time) repeated measures analysis of variance with Bonferroni's post hoc test. Differences in AUCs were analysed by ANOVA followed by Student–Newman–Keuls' post hoc test. All graphs were plotted using GraphPad Prism for Windows Version 5.00 (GraphPad, San Diego, CA).

3. Results In the present study, rats were selected to induce arthritis because rats develop a chronic swelling in multiple joints with influence on inflammatory cells, erosion of joint cartilage and bone destruction. Within 24 h of administering the CFA cardinal signs of inflammation, hyperalgesia, swelling, and hyperaemia were evident in all animals. Since measurement of vital signs of inflammation in both hind limbs is more practical and accurate than a visual grading system (Pearson and Wood, 1959) and also allows the effects of drug-treated groups to be expressed quantitatively, we measured the thickness of swollen ankle, hypersensitivity, and paw oedema of each rat joint. 3.1. Paw volume changes in adjuvant-induced arthritis IFA-injected Sprague-Dawley rats showed no significant paw volume change over the course of the study (Fig. 1A). Adjuvant injection resulted in progressive swelling of the injected (ipsilateral) hind paw that increased over time up to day 28 (Fig. 1A, C and E). The validity of the sensitivity of the model to anti-inflammatory drugs was demonstrated when the daily administration of aspirin suppressed both the maximal oedema effect (Fig. 1A) and the total oedema effect (Fig. 1B) in both the ipsilateral and contralateral paws. In the prophylactic study, all arthritic control rats showed acute inflammatory oedema at the ipsilateral (injected) paw around days 4–6 followed by a chronic polyarthritic phase which began around day 12–14 as previously described by Weichman (1989)

CFA Aspirin

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Total oedema response calculated as the area under the time course curve (AUC)

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Total oedema response calculated as the area under the time course curve (AUC)

D.D. Obiri et al. / Journal of Ethnopharmacology 152 (2014) 522–531

6000

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Fig. 1. Effect of Xylopia aethiopica extract on adjuvant-induced arthritis. Sprague-Dawley rats (200–250 g) were injected intraplantar with 100 µl of CFA or IFA into the right hind paw. Foot volume was measured before intraplantar injection of CFA/IFA and on alternate days up to 28 day. The oedema component of inflammation was monitored as the percentage change in paw volume (A, C and E). Total oedema induced was calculated as area under the time course curves, AUC (B, D and F). Drug effects were evaluated by comparing the maximal and total oedema responses attained during the 28 days in drug-treated groups with the corresponding values attained in drug vehicle-treated inflamed control groups. In the preventive (prophylactic) protocol (Top and Middle panels), drug-vehicle and either aspirin 100 mg kg
1 or XAE (100, 300 and 600 mg kg
1) was given orally 1 h before the induction of the arthritis and daily for 28 days while in the curative (therapeutic) protocol (Bottom panel), treatment commenced 14 days post arthritis induction. Data is presented as Mean ± s.e.m. (n=5). ***P ≤ 0.001; ** P ≤ 0.01; *P ≤ 0.05 compared to vehicle-treated group. Time course curves for paw volume were subjected to Two-way (treatment x time) repeated measure ANOVA followed by Bonferroni’s post hoc test). +++P ≤ 0.0005, ++P ≤ 0.005 prophylactically; ###P ≤ 0.0001 therapeutically; ns P ≥ 0.05 compared to vehicle-treated group (One-way ANOVA followed by Newman-Keul’s post hoc test). Arrow indicates point of extract administration in the therapeutic protocol.

and peaked on the day 24 (Fig. 1A and C). Total rat knee joint swellings induced over the 28 days were measured as the area under the time course curve, AUC (Fig. 1B and D). Treatment of

adjuvant-injected rats with XAE did not influence paw volume by day 8 (Fig. 1C). However, by day 14, extract treatments at all doses caused a decrease in primary paw inflammation compared with

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vehicle controls (po 0.05), and this reduction in primary paw volume continued to day 28 (p o0.05) (Fig. 1C). Xylopia aethiopica extract, XAE (100–600 mg kg
1) modified the time course curve significantly (P¼ 0.01, F¼7.11) (Fig. 1C) and reduced the oedema in the ipsilateral paw to 61.42 714.55%, 46.307 9.78% and 41.07 74.71% respectively (Fig. 1D). The progress of inflammatory oedema in the contralateral (non-injected) paw was evident from day 13 indicative of a systemic spread of the inflammation (Fig. 1A and C, inserts). In a separate experiment (therapeutic protocol), drug treatment commenced on day 14 after the start of the inflammation. Xylopia aethiopica, XAE (100–600 mg kg
1) significantly (P¼ 0.0005, F¼12.52) suppressed the spread of the oedema from the ipsilateral to the contralateral limb (Fig. 1E) and significantly reduced the total oedema to 60.93712.57%, 56.0779.24% and 42.9075.60% respectively (Fig. 1F). The progress of inflammatory oedema in the contralateral (non-injected) paw was evident from day 13 indicative of a systemic spread of the inflammation (Fig. 1E, insert). 3.2. Arthritis score The severity of the arthritis was measured in a blind manner on day 28 and scored by physical observation from photographs taken (pictures not shown). The non-arthritic (IFA) control rats recorded the lowest score and presented with no swellings compared to the CFA-treated model group which showed enormous erythema and swelling in both ipsilateral and contralateral paws and thus recorded the highest arthritic score in both prophylactic and therapeutic protocols (Fig. 2A and B). Administered prophylactically, XAE significantly (po0.05) suppressed the swellings in a dose-dependent manner thereby reducing the arthritic score in the ipsilateral paw (Fig. 2A). This effect of the extract was even more pronounced on the contralateral paws at 300 and 600 mg kg
1 with the arthritic score of the 100 mg kg
1 XAEtreated rats not significantly different from the aspirin-treated group (Fig. 2A). In the therapeutic regimen, however, XAE at 100 mg kg
1 did not significantly (p 40.05) reduce the arthritic score. The 300 and 600 mg kg
1 treatment caused a significant (p o0.05) and dosedependent reduction in the arthritic scores in both the ipsilateral and contralateral limbs (Fig. 2B).

3.3. X-ray radiography In the non-arthritic control (IFA)-group, no synovitis, pannus formation, focal cartilage or bone, erosion was seen in ankle joints (Plate 1A) while the arthritic model CFA-treated group showed observable signs of inflammation presented as massive inflammatory cell infiltrate, synovium hyperplasia, periarthritis and focal bone erosion in both the right and left hind limbs including the tissues surrounding the bones of the foot (Plate 1B). There are observable signs of inflammation at the metatarsal–phalangeal joint and the regions in-between the bones of the phalanges and the metatarsals. The rat hind limb shows bone enlargement with active osteophytosis, characterised by very thin trabeculae increasing from bone to connective tissue (1). The osteophytosis is marked on bone metaphysis and linked with lacunae (4). There are no observable joint spaces and some bone islets arise in the connective tissue (2), which is thickened and significantly enlarged (3). Eroding of the phalangeal bone is observed which was absent in the IFA group (Plate 1B). Inflammation spread to the fore limbs and slightly affected the carpals. The aspirin treated group showed no visible sign of bone and joint deformation but mild signs of inflamed tissues were observed (Plate 1C). In the prophylactic group XAE showed a dose-dependent suppression of inflammation and subsequent arthritic joint development (Plate 1D–F). Inflammation does not spread to affect tissues and bones of the fore limbs as was observed in the CFA-treated group. In the rats treated with 100 and 300 mg kg
1 XAE, there was early active osteophytosis in bone metaphysis with decreased bone density, moderate change of joint spaces (5) in a moderately enlarged and dense connective tissue (Plate 1D and E). Rats treated with 600 mg kg
1 XAE presented regions of minimal stabilised osteophytosis (6) (without newly formed trabecula) observable in the bone metaphysis and there is persistence of minor joint space alterations (7) (Plate 1F). In general, there are significantly reduced signs of bone deformation observed with the increase in doses of the Xylopia aethiopica extract given prophylactically. XAE 100 mg kg
1 presented mild eroding of the phalangeal bone comparable with the one observed in the aspirin-treated group. In the rats that were treated with XAE therapeutically, there was a dose-dependent prevention of inflammation when compared with the CFA group (Plate 1G–I). Inflammation does not spread to affect tissues and bones of the fore limbs as was observed in the CFA

5

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Fig. 2. Sprague-Dawley rats (200–250 g) were injected intraplantar with 100 ml of CFA or IFA into the right hind paw. Photographs of the hind limbs were taken on day 28 and the arthritic score physically determined as extent of erythema and oedema blindly scored on a scale of 0–4, with 0 being score for normal/uninjected control group. In the preventive (prophylactic) protocol (A), drug-vehicle and either aspirin 100 mg kg
1 or XAE (100, 300 and 600 mg kg
1) was given orally 1 h before the induction of the arthritis and daily for 28 days while in the curative (therapeutic) protocol (B), treatment commenced 14 days post arthritis induction. Data is presented as Mean 7 s.e.m. (n¼5). nnnPr 0.001; nnP r 0.01; nPr 0.05 compared to CFA-treated group.

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Plate 1. Sprague-Dawley rats (200–250 g) were injected intraplantar with 100 ml of CFA or IFA into the right hind paw. Rats were anaesthetised on 29th day by intraperitoneal injection of 50 mg kg
1 pentobarbitone sodium and X-ray radiographs taken.The severity of the joint and bone deformation was blindly scored according to the extent of osteoporosis, joint spaces, osteophytes and joint structure on a scale of 0–4, with 0 being score for normal/uninjected control group. In the preventive (prophylactic) protocol (Top and Middle panels), drug-vehicle and either aspirin 100 mg kg
1 or XAE (100, 300 and 600 mg kg
1) was given orally 1 h before the induction of the arthritis and daily for 28 days while in the curative (therapeutic) protocol (Bottom panel), treatment commenced 14 days post arthritis induction. Representative radiographs of rats treated with (A) IFA/non-arthritic control, (B) CFA/arthritic control, (C) Aspirin or (D-I) XAE 100, 300 and 600 mg kg
1. 1 ¼bone enlargement with active osteophytosis; 2¼ absence of observable joint spaces and some bone islets arising in the connective tissue; 3 ¼ thickened and significantly enlarged joint spaces; 4¼ osteophytosis marked on bone metaphysis and linked with lacunae; 5¼ early active osteophytosis in bone metaphysis; 6¼ regions of minimal stabilised osteophytosis; 7¼ bone metaphysis and persistence of minor joint space alterations; 8¼ zones of nominal stabilised osteophytosis.

treated group. Zones of nominal stabilised osteophytosis (8) (without newly formed trabecula) are visible in the bone metaphysis. Arthritic scores from the radiographs show that in both the contralateral and ipsilateral limbs, the model CFA group had highest score demonstrating severe bone enlargement with active osteophytosis in the bone metaphysis, osteolysis, focal areas of excessive bone resorption, subchondal erosion, and subluxation. As expected, the IFA control recorded the lowest score and presented with intact bones. When scored, prophylactically XAE-treated groups showed much enhanced inhibition of inflammation compared with the therapeutically treated groups. However, both treated groups showed a dosedependent inhibition of inflammation and arthritic joint when compared with the CFA and IFA treated groups. (Scores were not shown.) 3.4. Histopathology IFA-treated non-arthritic control rats had intact bone structure with no visible mononuclear cell infiltration nor synovial tissue

vasculature (Plate 2A) while the model CFA-treated rats showed mononuclear cell infiltration and vascularity in synovial tissues with characteristic redness, and pannus invasion of the subchondral bone (Plate 2B). In the aspirin-treated positive control group, rats showed highly vascularised bone tissues with characteristic redness, minimal mononuclear cell infiltration, and brittle bone structure, evident of degenerative arthritis (Plate 2C). When administered to the prophylactic group, XAE at 100 mg kg
1 caused mononuclear cell infiltration, and fluid infiltration of synovial cavity, and marked cavitation of bone structure, evident of osteoporosis (Plate 2D). In the 300 mg kg
1 XAE-treated rats, there was mononuclear cell infiltration, and pannus formation and invasion into the subchondral bone (Plate 2E). Remoulding bone structure with intact synovial lining and no visible mononuclear cell infiltration was observed in the 600 mg kg
1 XAE-treated group (Plate 2F). Therapeutically, fluid infiltration of synovial cavity, and marked cavitation of bone structure, evident of osteoclast activity was seen in the 100 mg kg
1 XAE-treated group (Plate 2G). There

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Plate 2. Sprague-Dawley rats (200–250 g) were injected intraplantar with 100 ml of CFA or IFA into the right hind paw. Rats were anaesthetised on 29th day by intraperitoneal injection of 50 mg kg
1 pentobarbitone sodium.The ipsilateral limbs were excised and fixed in Bouin's fluid, decalcified, sectioned and stained with haematoxylin and eosin and analysed microscopically. The histopathological change of joints was blindly graded on a score of 0–3. In the preventive (prophylactic) protocol (Top and Middle panels), drug-vehicle and either aspirin 100 mg kg
1 or XAE 100, 300 and 600 mg kg
1 was given orally 1 h before the induction of the arthritis and daily for 28 days while in the curative (therapeutic) protocol (Bottom panel), treatment commenced 14 days post arthritis induction. Representative histopathology images of rats treated with (A) IFA/non-arthritic control, (B) CFA/arthritic control, (C) Aspirin or (D-I) XAE 100, 300 and 600 mg kg
1.

was bone reformation structure with large marrow cavity and erythroblasts in the 300 mg kg
1 XAE-treated rats (Plate 2H). Re-calcified bone structures with osteoblastic excesses were evident in the 600 mg kg
1 XAE-treated rats (Plate 2I). When scored, prophylactically treated groups showed much enhanced inhibition of inflammation compared with the therapeutically‐treated group. However, both treated groups showed a dose-dependent reduction of inflammation and arthritic joint when compared with the CFA and IFA treated groups with an almost normal looking morphology of the synovium (scores not shown) 3.5. Haematological analysis Changes in the peripheral blood profile were observed after a period of injection of Complete Freund's adjuvant into the hind paw of rats. In the model CFA rats, haematological flusters, such as increase in WBC count, decrease in haemoglobin, RBC and haematocrit and increase in the ESR were observed. The administration of XAE both prophylactically and therapeutically, to the arthritic rats enhanced the HGB, RBC and HCT levels and reduced the WBC and ESR levels significantly when compared with the

arthritic control group. There were no significant differences in the haematological profile in the prophylactic and therapeutic protocols of the XAE treated rats (Table 1).

4. Discussion Adjuvant arthritis induced by heat-killed Mycobacterium tuberculosis cells is known to simulate the immunological and biochemical features of rheumatoid arthritis (Ramprasath et al., 2006). Auto-antigens that cross-react with Mycobacteria are implicated in the pathogenesis of rat adjuvant-induced arthritis and it is reported by Turull and Queralt (2000) that adjuvant arthritis appears as a consequence of an immune response to the cell wall of the Mycobacterium. When compared with normal rats, higher levels of IgG anti-Mycobacterium antibodies were observed in arthritic rats which also presented with delayed skin reactions induced by the soluble fraction of Mycobacterium. We found that at the doses used, XAE significantly decreased these humoral immune responses. At the same time, XAE treatment at all three doses assayed inhibited the delayed-type hypersensitivity seen in arthritic model animals. This significant finding suggests that XAE

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529

Table 1 Effect of XAE on haematological profile of SD rats. Treatment

WBC  109

HGB (g dL
1)

RBC  1012

HCT (%)

ESR (mm h
1)

CFA IFA Aspirin Prophylactic XAE (mg kg
1) 100 300 600 Therapeutic XAE (mg kg
1) 100 300 600

14.727 0.20 10.54 7 0.08n 10.707 0.10n

10.767 0.27 16.58 7 0.10n 14.50 7 0.25n

6.32 70.14 7.54 70.01n 7.62 70.21n

41.90 70.76 52.80 70.12n 55.76 70.30n

2.90 70.33 1.10 70.10n 2.30 70.12n

11.02 7 0.21n 10.647 0.41n 9.107 0.23n

12.50 7 0.11n 14.26 7 0.13n 14.90 7 0.22n

7.27 70.05n 7.42 70.11n 7.69 70.11n

49.18 70.22n 55.58 70.48n 54.12 70.84n

2.20 70.12n 1.40 70.19n 1.40 70.19n

12.30 7 0.06n 11.52 7 0.21n 10.32 7 0.08n

11.78 7 0.10n 12.20 7 0.09n 14.92 7 0.15n

6.84 70.03n 7.12 70.06n 7.91 70.17n

48.62 70.65n 51.98 70.26n 58.40 70.88n

2.20 70.12n 1.90 70.19n 1.50 70.16n

Sprague-Dawley rats (200–250 g) were injected intraplantar with 100 ml of CFA or IFA into the right hind paw. Blood sample collected from the jugular vein on day 29 was subjected to a full blood count with a blood analyser. The Biernacki reaction or eosinophil sedimentation rate (ESR) was done per the standard Westergren method. In the preventive (prophylactic) protocol drug-vehicle and either aspirin 100 mg kg
1 or XAE 100, 300 and 600 mg kg
1 was given orally 1 h before the induction of the arthritis and daily for 28 days while in the curative (therapeutic) protocol treatment commenced 14 days post arthritis induction. Data is presented as mean 7 s.e.m (n¼ 5). n

Pr 0.05 when compared to the arthritic control group (CFA).

might exert its effect through its influence on both the cellular and humoral immune response to the Mycobacterium in adjuvantinduced arthritic rats. RA is a chronic, systemic inflammatory disease characterised by chronic arthritis affecting several joints with accompanying synovial hyperplasia. Ultimately there is joint destruction, deformity and severe pain. Radiography, typically the first imaging study in evaluation for arthritis, offers true remission of disease and accurate evaluation of disease status (Kitamura et al., 2007). In the rat adjuvant-induced arthritis, there is increased bone resorption and decreased bone formation which results in bone loss (Findlay and Haynes, 2005; Makinen et al., 2007) and its progression is divided into three phases as in human rheumatoid arthritis. These phases start with the induction phase which has no evidence of synovitis, followed by early synovitis, and finally late synovitis with progressive joint destruction (Hoffmann et al., 1997). A good and classical anti-rheumatic agent should be able to block at least one or more of these phases. The aqueous ethanol extract of Xylopia aethiopica used in the work suppressed joint inflammation and synovitis. Regions of minimal stabilised osteophytosis were observable in the bone metaphysis and there was persistent minor joint space alterations in a dose-dependent manner in the extract-treated groups with reduced inflammation and joint deformation in both preventive and therapeutic protocols. Histopathology provides a noticeable morphological distinctiveness as a practical and unambiguous pathognomonic sign of RA (Soren, 1980). The histopathological analysis identified the ability of the bones to re-form upon treatment with XAE. Bone structures re-calcified upon treatment with the extract dosedependently. The aqueous ethanol extract of Xylopia aethiopica exhibited such therapeutic potential from the study results and is therefore consistent with earlier findings that the ability of a drug to suppress inflammation, synovitis and protect a joint is desired in rheumatoid arthritis therapy (Hoffmann et al., 1997; Sharma et al., 2004; Atzeni and Sarzi-Puttini, 2007). In RA, there is elevated WBC suggestive of inflammation as a consequence of infection; exercise and stress temporarily increase WBC (Braunwald et al., 2001). A lower haematocrit and RBC can be caused by a number of factors or conditions including rheumatoid arthritis (Braunwald et al., 2001). These observations were made in the negative control-group animals which were alleviated in the extract treated animals significantly. There is elevated Biernacki reaction in 95% cases of RA. Inflammation causes the body to produce higher proportions of fibrinogen in the blood which makes the red cells clump together, causing them to fall faster

than the healthy blood cells. Since inflammation can be caused by conditions other than arthritis, the Biernacki reaction is not a full diagnostic assessment of arthritis however, it does play a significant role in the diagnosis (Braunwald et al., 2001). The exact cause of RA reportedly remains unknown. However, the pro-inflammatory mechanism of RA is considered to be closely associated with progressive joint destruction in the progress of disease (Voog et al., 2003). At the molecular level, the array of cytokines manifested to play key roles in inflammatory development and bone erosion of RA include TNFα which is an autocrine stimulator and a potent paracrine inducer and thus increases the expression of other inflammatory mediators such as IL-1β, PGE2 and NO (Feldmann and Maini, 2008) and interleukin-6 (IL-6) to mediate and/or amplify their effects in peripheral organs (Cawthorn and Sethi, 2008). For example, while in vitro experiments have shown IL-1β potently induces matrix metalloproteinases generation and osteoclasts activation (Barksby et al., 2007) directly resulting in bone erosion evidences also exist for the potential degradation of cartilage and bone by TNFα (Zwerina et al., 2006). Therefore inhibiting the production and function of pro-inflammatory mediators is thought to be an effective method to treat RA. In support of this, Jin et al. (2010) report that drugs such as rituximab, etanacept, and tocilizumab, have been successfully approved in the clinical treatment of RA because they target specifically the TNFα receptor. After all, TNFα is designated a systemic marker of inflammation (MacNaul et al., 1990) and during the acute inflammatory process, its over expression is crucial to the induction of inflammatory genes and the recruitment and activation of host immune cells (Bhatia et al., 2005; Costa et al., 2013). The effect of XAE in suppressing the inflammation is also supported by the fact that a significant number of the constituent kaurane diterpenes, including kaurenoic acid, are known to inhibit iNOS, COX-2 and TNFα (Castrillo et al., 2001; Leung et al., 2005; Park et al., 2007; Sosa-Sequera et al., 2010). At the molecular level, the transcription factor NF-κB is inhibited by kauranes as reported by Castrillo et al. (2001). This impairs IκB kinase (IKK) activity as a result of the inhibition of NF-κB-inducing kinase (NIK). There is also a inhibition of signalling of the mitogen activated protein kinase, MAPK p38 and/or extracellular signal-regulated kinase 1/2 (ERK1/2). Activation of NF-κB has been shown to be a key component in the expression of genes involved in the production of inflammatory mediators and in RA active NF-κB plays a pivotal role in both the initiation and maintenance of chronic inflammation. Again, irrefutable documented evidences indicate that regulation of inflammatory genes such as TNFα and IL-6 is dependent on NF-κB (Collart et al., 1990).

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Taken together, it is consistent with our findings and not surprising therefore to see significant progressive decreases in thickness of both joints, and suppression of paw oedema in rats treated with XAE compared to vehicle-treated diseased rats. These marked inhibitions in swelling were observed in the time-course of the pathology of adjuvant-induced arthritis in both ipsilateral and the contralateral limbs when XAE was administered orally in both the prophylactic and therapeutic regimens. This mechanism possibly explains, at least in part, the observed anti-arthritic effect of XAE which contains at least two kaurenes: kaurenoic acid and xylopic acid. We have subsequently isolated the principal constituent, xylopic acid and started investigation into its molecular mechanism of action and its effect especially on the NF-κB pathway and consequently on serum TNFα and IL-6 levels.

5. Conclusion The aqueous ethanol fruit extract of Xylopia aethiopica is an effective anti-arthritic agent experimentally and holds prospect in future rheumatoid arthritis treatment. In conclusion, we have verified that XAE suppressed the joint inflammation and destruction in adjuvant arthritic rats. We are confident that our data provide mechanistic evidence for anti-arthritic appliance of XAE as a promising candidate for novel therapeutic agent of RA.

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