EVALUATION OF THE ANTI-ARTHRITIC EFFECT OF STERCULIA TRAGACANTHA (LINDL.) LEAF EXTRACT IN RATS

Sterculia tragacantha leaves for years have been used by traditional hea lers in eastern Nigeria in the treatment of arthritis, edema, gout, whitlow and cold. The ai m of this study was to evaluate the folkloric claim s of Sterculia tragacantha leaf extract in relieving arthritic conditions. Th e effects of Sterculia Tragacantha methanol leaf Extract (STEX) on formaldehyde and ad juvant-induced arthritis were studied in rats. Paw thickness, White Blood Cell Count (WBC) count, Pack ed Cell Volume (PCV), Haemoglobin Concentration (HB), Erythrocyte Sedimentation Rate (ESR), lipid p eroxidation, Super Oxide Dismutase (SOD) activity a nd catalase activity were studied post induction of ar thritis. In both formaldehyde and adjuvant-induced arthritis studies, mean paw thickness in animals given 300 mg kg STEX was significantly (p<0.05) lower on days 7 and 14 compared to that of normal saline group. Mea n WBC of 100 and 300 mg kg v STEX groups were significantly (p<0.05) lower than that of normal sa ine group. Mean ESR of normal saline and 100 mg kg −1 STEX groups were significantly (p<0.05) faster than ESR of 300 mg kg −1 group. Mean MDA level of 300 mg kg STEX group was similar to that of non-arthritic gr oup while mean SOD levels of 300 and 100 mg kg −1 STEX groups were significantly (p<0.05) higher than that of normal saline group. Mean catalase level o f 100 and 300 mg kg −1 STEX groups were significantly (p<0.05) higher com pared to that of normal saline group. These results show that STEX exhibited potent antiarthritic activity.


INTRODUCTION
Joint diseases are broadly classified as inflammatory or non-inflammatory disorders (Halliwell and Gorman, 1989). Degenerative joint disease (or osteoarthritis) is a non-inflammatory joint disorder while inflammatory diseases of the joint include feline progressive polyarthritis, lupus polyarthritis, idiopathic non erosive arthritis and rheumatoid arthritis (Todhunter and Johnston, 2003;Hegen et al., 2008). Rheumatoid Arthritis (RA) is a chronic auto immune-mediated disease which affects humans and animals (Kahn, 2005;Kaur et al., 2012).
This joint disorder also affects tissues and organs such as the heart, lungs, eye and neuromuscular system (Mahajan et al., 2010). In the joint, RA is characterized by profuse inflammatory reaction in the synovial membrane and subchondral bone which results in progressive erosion of articular cartilage and synovitis (Hegen et al., 2008;Kaur et al., 2012). In advanced cases, ankylosis, subluxation, soft tissue destruction, disuse osteoporosis and pain may be noticed (Halliwell and Gorman, 1989;Goldring and Goldring, 2006).
There is no known cure for RA but several drugs such as anti-inflammatory and disease modifying anti-

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AJPT rheumatoid drugs are used in mono or combination therapies to inhibit the disease process (Makinen et al., 2007;Zhao et al., 2006;Mottonen et al., 2006). However, prolonged use of these drugs is associated with deleterious side effects such as gastric ulceration, haemorrhage, anemia and kidney dysfunction (Lin et al., 2006;Buhroo and Baba, 2006;Kyei et al., 2012). Thus in recent times, researches have been directed towards the use of biologics (Ruggiero et al., 2009) and plantderived drugs in the treatment of RA (Woode et al., 2009;Kaithwas and Majumdar, 2010).
Several plants such as aloe barbadensis (Josheph and Raj, 2010), actaea racemosa (Bang et al., 2009), caloptropis procera (Vaidya, 2006), parlisota hirsuta (Woode et al., 2009) and linum usitatissimum (Kaithwas and Majumdar, 2010) have shown promising treatment option for rheumatoid arthritis. Traditionally, decoctions made from leaves, bark and seeds of Sterculia tragacantha (family: Sterculiaceae) tree are used in the treatment of arthritis, edema, gout and whitlow (Iwu, 1993). Recently the methanol extract of S. tragacantha extract was reported to possess analgesic, anti-inflammatory and in vitro anti-oxidant effects (Udegbunam et al., 2011). Therefore bearing in mind the antioxidant, analgesic and anti-inflammatory effects of S. tragacantha and its use in traditional medicine in the management of arthritis, we considered it worthwhile to investigate its anti-arthritic effect.

Plant Material
Fresh leaves of S. tragacantha were collected in February, 2012 and authenticated by a taxonomist at the Biodiversity Development Centre (BDCP) Nsukka. The leaves were air dried, pulverized and cold macerated in 80% methanol for 48 h at 37°C with intermittent shaking every 2 h. After 48 h the extract was filtered and evaporated to dryness in a rotary evaporator to obtain an extract (yield: 12.0%). Previously, phytochemical analysis of methanol extract of this plant revealed the presence of alkaloids, flavonoids, tannins, glycosides and saponins (Udegbunam et al., 2011). Doses used in this study were selected based on previous acute toxicity and anti-inflammatory studies (Udegbunam et al., 2011).

Formaldehyde-Induced Arthritis
Non-immunological arthritis was induced in four groups (n = 5) of rats by sub plantar injection of 0.1 mL freshly prepared 2.5% formaldehyde (Seyle, 1949) on day 1 and repeated on day 3. Groups 1 and 2 received 100 and 300 mg kg −1 body weight (b.w) S. Tragacantha methanol leaf Extract (STEX) orally (p.o) while groups 3 and 4 received 5 mg kg −1 b.w piroxicam (i.m) and normal saline (1 mg kg −1 , p.o) 1 h before arthritis induction. The extract and piroxicam were administered once daily for a period of 10 days. The paw thicknesses of rats were measured on days 0, 3, 5, 7 and 10 using a venire caliper. The edema component of arthritis was estimated by calculating the difference between day 0 paw thicknesses and paw thicknesses at the various time points. Blood samples were collected from retro orbital plexus on days 0, 3, 7 and 10 for total white blood cell count (Bain et al., 2012). On day 10, rats were euthanized and paw tissues collected to determine Malondialdehyde (MDA) level as well as Superoxide Dismutase (SOD) and Catalase (CAT) activities in rat paw tissues. MDA was determined as described by Ohkawa et al. (1979), SOD activity was estimated using the procedure of Sun et al. (1988) while CAT was assayed as described by Sinha (1972). MDA, SOD and CAT levels in paw tissues of a non-arthritic/normal group (group 5) were also studied and compared with those of the arthritic/treatment groups.

Freund Adjuvant (CFA) Induced Arthritis
Immunological arthritis was induced in rats according to the method of Newbould (1963). The left foot pad of each rat was injected subcutaneously with 0.05 ml Freund's adjuvant complete (Sigma Aldrich, Germany). Animals in groups 1, 2, 3 and 4 received 100 mg kg −1 b.w STEX, 300 mg kg −1 b.w STEX, 5 mg kg −1 b.w piroxicam and 1 mL kg −1 b.w normal saline 24 h respectively before adjuvant injection. The treatments were re-administered once daily for 28 days. The left paw and tarso-metatarsal joint thicknesses were measured on days 3, 7, 14, 21 and 28 post induction of arthritis using a venire caliper. The edema component of arthritis was estimated by calculating the difference between day 0 paw thicknesses and paw thicknesses at the various time points. On day 28, blood was collected to determine the Packed Cell Volumes (PCV), Haemoglobin Concentrations (HB), red Blood Cell Counts (RBC), White Blood Cell counts (WBC) and Erythrocyte Sedimentation Rates (ESR) of rats. PCV was determined by the microhaematocrit method, HB was determined by the cyanmethaemoglobin method, RBC

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AJPT and WBC were determined by the haemocytometer method while ESR was determined using the Westergren method as described by Bain et al. (2012). PCV, HB, RBC, WBC and ESR of non-arthritic/normal group (group 5) were also studied and compared with those of the arthritic/treatment groups.

Statistical Analysis
Data obtained were compared between groups using one way Analysis of Variance (ANOVA). Duncan multiple range tests were used to test for significance differences between means at probability less than 0.05.

Formaldehyde-Induced Arthritis
The effect of S. tragacantha extract on formaldehyde induced arthritis is summarized in Table 1. The results showed that mean paw thickness of 300 and 100 mg kg −1 STEX groups were significantly (p<0.05) lower than that of normal saline group. Mean WBC of 100 and 300 mg kg −1 STEX groups were significantly (p<0.05) lower than that of normal saline group ( Table 2). Mean MDA levels of normal saline and 100 mg kg −1 STEX groups were significantly (p<0.05) higher than MDA levels of non-arthritic and 300 mg kg −1 STEX groups. MDA levels of 300 mg kg −1 STEX and piroxicam groups were similar to that of non-arthritic group ( Table 3). Mean SOD levels of 300 and 100 mg kg −1 STEX groups were significantly (p<0.05) higher than SOD level of normal saline group. Mean catalase level of 100 and 300 mg kg −1 STEX groups were significantly (p<0.05) higher compared to that of normal saline group ( Table 3).

DISCUSSION
The inflammatory process is a physiologic response of a living organism to factors such as infection, trauma or immunological mechanisms (Tanas et al., 2010). This process is initiated by the host to eliminate irritants and to set the stage for tissue repair (Bhitre et al., 2008). In this study, arthritis, a chronic joint inflammatory disease, was induced in rats using formaldehyde and Complete Freund's Adjuvant (CFA). Formaldehyde injection elicits localized inflammation and pain in the early phase followed subsequently by a phase of tissue mediated response (Aceto and Cowan, 1991). This late phase produces proliferative joint inflammation leading to articular changes similar to those seen in rheumatoid arthritis (Okoli et al., 2008). CFA on the other hand initiates progressive joint destructi on characterized by synovitis, polyarthritis and systemic infl ammation (Woode et al., 2009). Thus, formaldehyde and CFAinduced arthritis are commonly used experimental models

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AJPT for preclinical screening of non-steroidal anti-inflammatory drugs, disease modifying anti-rheumatoid drugs and plant extracts for anti-arthritic effect (Woode et al., 2009). The results obtained in this study showed that STEX significantly suppressed formaldehyde and CFA-induced arthritis as shown by the significantly lesser paw and joint thickness in 300 mg kg −1 STEX group post arthritis induction. However, measurement of paw and joint thickness gives only an indication of edematous changes in these regions (Woode et al., 2009), therefore to correlate the edematous changes with the local biochemical changes, tissue MDA, SOD and catalase level activities in rat paw were measured during formaldehyde-induced arthritis.
Phagocytes such as macrophages and neutrophils which invade inflamed tissues generate reactive oxygen species (Valko et al., 2006). ROS apart from being defensive, when in excess deregulate cellular function causing oxidative damage which worsens inflammation (Wu et al., 2006;Tanas et al., 2010). Cells contain a number of anti-oxidants such as superoxide dismutase, catalase and glutathione peroxidase which prevent the damage caused by ROS (Weydert and Cullen, 2010). SOD converts superoxide radical to hydrogen peroxide and oxygen while catalase decomposes hydrogen peroxide into water (Weydert and Cullen, 2010). In this study, SOD and catalase levels of normal saline and 100 mg kg −1 groups were significantly lower than that of non-arthritic rats. Earlier studies have shown that SOD and catalase levels decreased in chronic inflammatory states (Halici et al., 2007;Wu et al., 2006;Govindarajan et al., 2007). Thus we can infer that the presence of formaldehyde in the tissues stimulated a profuse production of ROS which significantly overwhelmed the antioxidant system in rat paw tissues leading to the decrease in SOD level. However, SOD and catalase levels in the paws of rats treated with 300 mg kg −1 STEX were almost similar to that of non-arthritic rats suggesting that severe paw inflammation was attenuated by administration of this treatment. The finding may be linked to the antiinflammatory and free radical scavenging effects of STEX (Udegbunam et al., 2011).
The level of MDA in the tissue is considered a measure of lipid peroxidation which is linked to the production of superoxide radical (Karatas et al., 2003). Increased level of MDA as seen in normal saline group was an indication that the presence of formaldehyde in the tissues stimulated profuse production of free radicals. Increased lipid peroxidation in rat paw tissues following injection of irritants such as carrageenan has been reported (Tanas et al., 2010). Furthermore, the lower MDA level in the 300 mg kg −1 STEX and piroxicam group suggests that both treatments ameliorated the inflammatory process thus dampening the production of free radicals.
In formaldehyde and CFA induced-arthritis, WBC of the normal saline treated rats was higher than those of the other treatment groups. The increase in WBC in all the groups followed the same pattern as the degree of paw inflammation. Previously, leucocytosis and neutrophilia characterized adjuvant-induced arthritis in rats (Franch et al., 1994). White blood cells are important components of the host defense system (Mahgoub et al., 2008) thus the increased WBC seen in this study can be attributed to systemic response of the rats to paw inflammation induced by formaldehyde and Freund adjuvant (Franch et al., 1994). Furthermore, lower WBC in the 300 mg kg −1 STEX group suggests that the extract showed potent antiarthritic effect given that elevated WBC are associated with active inflammation (Kyei, 2012).
PCV, HB and RBC of normal saline and STEX groups were not significantly different from those of non-arthritic rats. Earlier, Kyei (2012) reported that RBC and HB of rats were not affected post induction of arthritis. However, this author reported a significant increase in ESR during arthritis. Our finding also showed that ESR was significantly faster in normal saline and in 100 mg kg −1 STEX groups but was slower in 300 mg kg −1 STEX group. Therefore, since proteins produced during inflammation cause erythrocytes to stack up in a group leading to faster settling (Kyei, 2012), the elevation of ESR observed in 100 mg kg −1 STEX group showed the presence of high quantity of inflammatory proteins in circulation while the near normal ESR in 300 mg kg −1 STEX group points to the fact that inflammation was less severe in this group.

CONCLUSION
This study showed that daily administration of 300 mg kg −1 STEX significantly ameliorated the arthritic process as shown by lesser local (paw edema and tissue anti-oxidant activities) and systemic (WBC and ESR) changes in rats treated with this dose of STEX. No adverse haematologic effects were noted following the use of both doses of STEX. Therefore we conclude that S. tragacantha can serve as a good anti-arthritic agent. Further works are on-going to isolate the bio-active compound in this extract and determine its mechanism(s) of action.