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Md Imaduddin*, Chandrashekhar VM, Firasat Ali, Gudasi KB, Parvinder Singh.
Department of Pharmacology, HSK College of Pharmacy, Bagalkot–587101, Karnataka, India.

ORIGINAL RESEARCH ARTICLE
Volume 2, Issue 3, Page 136-141, September-December 2014.

Article history
Received: 5 November 2014
Revised: 25 November 2014
Accepted: 30 November 2014
Early view: 15 December 2014

*Author for correspondence
E-mail: imadpharma111@gmail.com

ABSTRACT

Background: In the field of medicinal chemistry quinazolinone has gained unique place due to anti-microbial, anti-inflammatory, analgesic, anti-convulsant, anti-hypertensive, diuretic and anti-viral activity. 3-methoxysalicylaldehyde-2-aminobenzoylhydrazone (AQF) was complexed with transition metal nickel (Ni) and used to evaluate anti-arthritic activity in Freund’s complete adjuvant (FCA) induced arthritis in Sprague–Dawley (SD) female rats.
Materials and methods: The anti-arthritic activity of Ni-AQF (5mg/kg and 10mg/kg) was evaluated in rats subjected to Adjuvant Arthritis (AA) by the sub-plantar injection of 0.1ml of FCA. The anti-arthritic activity of Ni-AQF was assessed by determining the Paw oedema volume, vascular permeability test, histamine level in blood. It was also determined by radiographic analysis, Histopathological assessment of arthritis induced joints.
Results: Animals treated with Ni-AQF showed significant reduced paw oedema, release of histamine into the blood, redness, swelling, and erythema at ankle joints. Furthermore Ni-AQF treatment showed reduced infiltration of lymphocytes, swelling deformities and joint spaces. Histopathological assessment of rats treated with Ni-AQF showed mild Proliferation and infiltration of mononuclear cells, mildsub-chondral bone erosion and superficial cartilage damage as compared to control group.
Conclusions: These results suggested the protective role of Ni-AQFin FCA induced chronic arthritis model. In our present study, Diclofenac sodium was used as a reference standard, which is one of the familiar non-Steroidal anti-inflammatory drugs (NSAID’s) for the initial treatment for rheumatoidarthritis (RA). Further, studies are required to elucidate the detail mechanism of action of this agent at molecular level to explore the therapeutic benefits.

Keywords: Rheumatoid arthritis, paw oedema, inflammation, 3-methoxysalicylaldehyde-2-aminobenzoylhydrazone.

INTRODUCTION

Rheumatoid arthritis (RA), a chronic inflammatory disease of unknown etiology, is characterized by hyperplasia and chronic inflammation of the synovial membranes that invade deeply into the articular cartilage and bone (Firestein, 1996). Proliferative fibroblast like synoviocytes play crucial role in both the progression of inflammation and joint damage because they produce a great amount of pro-inflammatory mediators such as matrix metalloproteinase, interleukin(IL)-6, IL-1β, and prostaglandin E2 (PGE2)(Mor et al., 2005). Management of RA has relied on symptomatic treatment with a variety of anti-inflammatory drugs, toxic immunosuppressant’s, corticosteroids, viscosupplements (for joints) and a series of new biological monoclonal antibodies (anti-TNF, anti-IL-1Ra, anti-CD 20, anti-IL-2, IL-4). The conventionally user therapies for inflammation, NSAID’s have very important role in managing pain and inflammatory conditions (Davies et al., 2000) which are the discouraging profile of side effects (James and Hawkey, 2003). It would, therefore, be highly desirable to find less toxic alternatives to treat RA.
Quinazolinone is an exciting motif as regards its pharmacological importance and is a building block for about 150 naturally occurring alkaloids (Mhaske and Argade, 2006). Diproqualone- a derivative quinazoline-4-one which has been used primarily for the treatment of inflammatory pain associated with osteoarthritis and RA (Audeval et al., 1988). Quinazolinones occupy a promising section in the anti-cancer market because of their specificity. Therefore synthesis, physico-chemical characterization and anti-microbial activity of title compound and various transition metal complexes has been carried out and published in journal of molecular structure (Badiger et al., 2012). In future we can expect novel drugs from this scaffold which would be more specific for various ailments. The present work was, therefore, conducted to assess anti-arthritic activity of Ni-AQF in FCA induced arthritis in SD female rats.

MATERIALS AND METHODS

Synthesis and characterization of Ni-AQF
According to our earlier publication as mentioned above 3-methoxysalicylaldehyde-2-aminobenzoylhydrazone was synthesized by temperature controlled condensation of 2-aminobenzoylhydrazide and 3-methoxysalicylaldehyde which was characterized by various spectroscopic methods like NMR, UV-Vis, IR and Mass and also by thermal and physico-chemical methods.
Dose selection
Dose selection of the test drug, Ni-AQF was based on the acute toxicity test. The female Swiss-mice (25-30 g) were used and acute toxicity was studied according to the OECD guidelines-425. Limit dose of 2000 mg/kg (p.o.) and 550 mg/kg (p.o.) was administered and followed by behavioral and toxicological signs were observed for 4 hrs and monitored up to 14 days and 100% mortality was occurred with higher limit dose. LD50 was calculated with the help of AOT software. According to the results of acute toxicity test, the doses of 5 mg/kg body weight (BW) and 10 mg/kg BW were chosen for the experiment.
Animals
Anti-arthritic activity was carried on SD female rats (150-200 g). Rats were obtained from the central animal house of HSK College of Pharmacy and Research Centre, Bagalkot, KA, India. All animals were kept under standard husbandry conditions (Temp. 22-28 °C; Relative humidity 65±10%) for 12hr dark and 12hr light cycle respectively in standard propylene cages. The animals were fed with standard food (Pranav Agro Industries, Sangli, Maharashtra, India) and water ad libitum. All the experiments were conducted in accordance with direction of Institutional Animals Ethics Committee (HSKCP/IAEC, Clear/2010-11/1-14). All efforts were made to minimize distress and the number of animals used.
Induction of AA and treatments
The FCA induced chronic anti-arthritic activity of Ni-AQF (5mg/kg and 10mg/kg) was carried out and female SD rats (150-200g) were divided into 4 groups, contains 8 animals in each group
Group I. Control group received vehicle (10 % v/v tween 80; p.o.) + FCA induced changes in rat paw oedema
Group II. Effect of diclofenac (10 mg/kg; p.o.) on FCA induced changes in rat paw oedema
Group III. Effect of Ni-AQF (5 mg/kg; p.o.) on FCA induced changes in rat paw oedema
Group IV. Effect of Ni-AQF (10 mg/kg; p.o.) on FCA induced changes in rat paw oedema
AA was induced by the sub-plantar injection of 0.1ml of FCA on day zero (kalia et al., 2007). Everyday animals were carefully and thoroughly inspected, by examining the affected paw and general status. In FCA induced animals, the sub-plantar injection of FCA produces local oedema after few hours with a progressive increase reaching its maximum up to 28th day.
Effect of Ni-AQFon FCA induced paw oedema
On 0th, 7th, 14th, 21stand 28thday the paw-oedema volume of each rat was measured thoroughly in all groups by using digital Plethysmometer (7140 UGO Basile). The percentage inhibition of paw oedema volume of each treated groups is calculated by the equation,
Percentage edema inhibition (EI), % = (Vc – Vt / Vc) 100
Where, Vt = Mean paw volume of each treated group
Vc= Mean paw volume of control group
Effect of Ni-AQF on vascular permeability test in FCA induced arthritis in rats
Evan’s blue 50 mg/kg of BW was administered via the jugular vein into the anaesthetized rat. After 4hr of Evans blue administration, each rat was sacrificed by anesthetic ether. The anterior and posterior synovial capsules and fat pad were dissected from each knee joint. The tissue obtained from each knee was then weighed, and amount of Evans blue in the sample was estimated by extracting the dye by following procedure.
This entailed cutting the capsule in to smaller pieces and mixing them with acetone in 1% NaSO4 in the ratio of 7:3. The samples were shaken gently and continuously for 24 h at room temperature. Each preparation was centrifuged for 10 min at 2000 rpm and 2 ml of supernatant was separated for measurement of absorbance at 620 nm using UV- spectrophotometer (UV- 1601, Shimadzu Corporation, and Kyoto, Japan). The amount of dye recovered content of vascular permeability was calculated by extrapolating with standard curve prepared with different concentrations of Evans blue solution. (Figure 1A) The percentage of inhibition of joint infiltration was calculated by the equation, (Franchiset al., 2004).
Percentage inhibition % = (Vc-Vt / Vc) x 100
Where, Vt = Mean Evans blue joint infiltration of treated groups
Vc = Mean Evans blue joint infiltration of control group.
Effect of Ni-AQFon release of histamine from blood in FCA induced arthritis in rats
The 5.0 ml of blood was taken from cardiac puncture of a rat heart and mixed with 6.0 mg of ammonium oxalate in a test tube then add 4.5 ml of distilled water and 0.5 ml of 10-12 N perchloric acid (HClO4) and it is kept at room temperature for 10 minutes. For the above mixture add 9 volumes of 0.4NHClO4 then homogenate in a motor-driven glass homogenizer. The homogenate is allowed to stand for 10 minutes and is then centrifuged. The supernatant fluid 4.0 ml was shaken with 0.5 ml of 5N NaOH, 1.5 gm of solid sodium chloride and 10ml of n-butanol. The tube is shaken for 5 minutes to extract the histamine into butanol. After centrifugation aqueous phase is removed by aspiration. The organic phase is then shaken for about 1 minute with 5.0ml of Salt-saturated 0.1N NaOH. This wash gives removal of residual amounts of histidine which may be present. After centrifugation 8.0 ml of butanol phase (organic phase) is taken, to this add 4.5ml of 0.1NHCl and 15.0 ml of n-heptane. Shake for 1min. aqueous phase is collected after the centrifugation and is used for the assay fluorimetrically (Shore et al., 1959). Then, 2 ml of aliquot was taken, mix 0.1 ml of O-phthaldehyde (OPT) reagent and then added 0.2 ml of 3N HCl. The fluorescence of acidified solution was stable for at least 90 min. The fluorescence intensity was proportionally to histamine concentration over the range of 0.005 to 0.5 μg/ml. Standard curve was prepared by preparation of 2.0 ml aliquots with standard histamine. The histamine concentration of treated groups was determined by extrapolating with the standard graph plotted with different concentrations of histamine against fluorescence (Figure 1B) (Chitme et al., 2010).
Effect of Ni-AQFon radiographic analysis in FCA induced chronic arthritis in rats
On 28th day, animals were anesthetized with Ketamine hydrochloride (45 mg/kg). Radiographs of the FCA injected joints were taken with an x-ray (Model no DX-300., Pune, India). Rats were placed on a radiographic plate at a distance of 75 cm from the X-ray source and the machine was operated at 46 kV peak, 4 mA and exposure time was 0.8 sec. The X-ray image of the FCA injected joints of each rat was evaluated for radiographic changes (Kalpeshet al., 2011).
Effect of Ni-AQFon histopathological assessment in FCA induced arthritis in rats
The rats were anaesthetized and exsanguinated and their knee joints are dissected, freed from muscles and fixed in 10% formalin (Franchis et al, 2004). The joints are decalcified, embedded in wax, sectioned and stained with haematoxylin and eosin. Histological analysis was carried out by a single observer, focusing on polymorph nuclear cell infiltration, tissue proliferation and cartilage erosions. The severity of the lesions was given scores: 0 = no change, 1 = mild change, 2 = moderate change and 4 = marked change
Statistical analysis
The obtained results were analyzed using the Graph Pad Prism 5.0 and expressed as Mean ± SEM. Statistical differences between groups were calculated by one-way ANOVA followed by Dunnett’s multiple comparison test.

Figure 1A. Standard curve prepared with different concentrations of Evans blue solution.
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Figure 1B.. Standard curve prepared for the estimation of release of histamine in blood by flourimeter.
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RESULTS

Effect of Ni-AQF on FCA induced paw oedema
In FCA induced chronic arthritis model, control group animals showed increased paw oedema gradually up to 28th day. The test compounds Ni-AQF showed significant reduced right paw oedema from 14th day onwards and up to 28th day as compared to control group. The results were summarized in Fig. 2.

Figure 2. Effect of Ni-AQF on FCA induced paw oedema.
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Effect of Ni-AQFon release of histamine from blood in FCA induced arthritis in rats
The histamine release in blood was estimated. In control group animals showed significant (p<0.001) elevated histamine content in blood was observed. In contrast, the standard diclofenac and test compounds Ni-AQF showed significant (P < 0.001) reduced release of histamine into the blood was observed. The results were presented in the Fig. 3A.

Figure 3A. Effect of Ni-AQF on release of histamine from blood (3A) and effect of Ni-AQF on vascular permeability test.
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Figure 3B. Figure 3B. Effect of Ni-AQF on vascular permeability test
(3B) in FCA induced arthritis in rats.
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Effect of Ni-AQFon Vascular permeability test in FCA induced arthritis in rats
In the present study control group has showed significant increased infiltration of lymphocytes, indicated by increased Evans blue extravasation which is due to increased endothelial gap of vascular components at knee joint of rats. Treated groups showed significant reduced infiltration of lymphocytes, diclofenac (p<0.001), Ni-AQF 10 mg/kg (p<0.001) as compared to control group. In treated groups, the percentage inhibition of joint infiltration is 00.00% and 50.45% in Ni-AQF of 5mg/kg and 10mg/kg respectively. The significant decrease in extravasation of dye in Ni-AQF is due to reduced endothelial gap of vascular component at knee joints of rats. The results were presented in Fig. 3B.

Figure 4. Radiographic assessment of FCA induced arthritis in rats. Radiographs taken on 28th day after FCA injection(A) control; shows severe swelling, deformity and increased joint spaces(B) Diclofenac 10 mg/kg treated animals showing significant reduction in soft tissue swelling and bone erosive changes, (C) Ni-AQF 5 mg/kg, (D)Ni-AQF 10 mg/kg shows significant reduction in swelling deformities and decrease in joint spaces..
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Effect of Ni-AQFon radiographic analysis in FCA induced arthritis in rats
FCA injected group of animals shown deformation and abnormality in toes. Treatment with diclofenac 10 mg/kg, Ni-AQF (5 mg/kg and 10 mg/kg) shown to reduce swelling deformities and decrease in joint spaces (Figure 4).
Effect of Ni-AQFon histopathological assessment in FCA induced arthritis in rats
The extent of rat paw pathological conditions were given scores (Table 1). Paws were observed on Light microscopy 10-x. Paw joint tissue was fixed in 10% formaldehyde and 5-μm paraffin sections were stained with hematoxylin and eosin. Control group showed destruction of cartilage and sub-chondral bone, disorganization of the joint space and replacement with mononuclear cells and fiber thickening, increased lymphocytes and plasma cells. Treatment with diclofenac 10 mg/kg, Ni-AQF (5mg/kg and 10 mg/kg) showed mild Proliferation and infiltration of mononuclear cells, mild sub-chondral bone erosion and superficial cartilage damage as compared to control group.

Figure 5. Histopathological assessment of FCA induced arthritis in rats(A) control; shows destruction of cartilage, replacement with mononuclear cells, increased lymphocytes and plasma cells (B) Diclofenac 10 mg/kg, Ni-AQF 5 mg/kg (C), Ni-AQF 10 mg/kg treated animals shows mild Proliferation and infiltration of mononuclear cells, mild sub-chondral bone erosion and superficial cartilage damage.
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Table1. Effect of Ni-AQF on histopathological assessment in FCA induced arthritis in rats.
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DISCUSSION

The chronic inflammation involves the release of various inflammatory mediators like cytokines (IL-1α and TNF-α), granulocyte monocytes colony stimulating factor (GM-CSF), platelet derived growth factor (PGDF) and others. These mediators are responsible for the pain, destruction of cartilage and leads to severe disability. Paw swelling is one of the major factors in assessing the degree of inflammation and efficacy of the drugs. AA is non-specific immune response within the joint can also result in inflammatory and erosive disease (Paul, 2008).
Paw swelling is an index of measuring the anti-arthritic activity of various drugs and it is employed here to determine the activity of Ni-AQF quinazolinone metal complex. Reference standard diclofenac sodium, Ni-AQF administered groups showed marked reduction in paw volume when compared with the arthritic control group by inhibiting the release of inflammatory mediators. As inflammation is progressed, a more diffused demineralization developed in the extremities (Begum and Sadique, 1988).
The swelling of joint knees due to FCA induced arthritics, causes blood vascular permeability within 28 days of administration. Evans blue extravasations method is used to assess for plasma protein extravagations in the rat knee joint, because Evans blue has high binding affinity to plasma proteins. Normally, large plasma proteins and bound Evans blue dye cannot pass through the endothelial gaps and therefore gets restricted in the vascular component. Endothelial cells undergo activation, expressing adhesion molecules and presenting chemokine’s, leading to enlargement of endothelial gaps as a result plasma protein and Evans blue dye complex can escape to the interstitial tissues. The measurement of the amount of Evans blue in the synovial capsule can provide us an index of the relative vascular permeability. The decreased extravasations reported for Ni-AQF and diclofenac was due to decreased endothelial gaps which are caused by decreased expression of adhesion molecules. The extraction of histamine into n-butanol from alkalinized perchloric acid tissue extracts, return of the histamine to an aqueous solution and condensation with OPT to yield a product with strong and stable fluorescence which is measured in a spectrofluorimeter. Preformed histamine is present in mast cell granules and gets released by mast cell degranulation in the inflammatory conditions. Thus released histamine gets involve in the immune reactions, which involves binding of antibodies to mast cells and releases neuropeptides and cytokines like IL-1, IL-6, IL-18 that plays an important role in the RA (William, 1996; Wood et al., 1992). The quinazolinone derivative had shown the decreased level of histamine, release in blood and also observed decreased oedema, redness and swelling.
Radiographic changes in RA conditions are useful diagnostic measures which indicate the severity of the disease. The radiographic (x-ray) analysis of the joints in the arthritis Control group showed the soft tissue swelling, bone deformities, bone erosive changes and bone resorption. This indicates the confirmation of arthritis. The significant reduction of above pathological changes was observed in Ni-AQF and standard treated group as compared to control group.
Histopathological analyses revealed that, in relation to the development of inflammatory and arthritic lesions in the paws, most of the FCA rats experienced progressive cartilage destruction and bone erosion, finally leading to complete ankylosis and malformation of the joints In contrast, treatments with Ni-AQF not only showed significant reduction of cellular infiltration, joint space narrowing, synovial hyperplasia, and pannus formation, but also markedly protected the affected joints against cartilage destruction and bone erosion.

CONCLUSION

In conclusion, Ni-AQF act as a therapeutic agent for the treatment of AA induced by FCA in SD female rats by suppressing the paw oedema, soft tissue swelling, vascular permeability, bone erosion and cartilage destruction. Further, studies are required to elucidate the detail mechanism of action of these agents at molecular level to explore the therapeutic benefits and in the near future, we may expect a new therapeutic option for the treatment of arthritis.

CONFLICT OF INTEREST
Authors report no conflict of interest. Authors alone are responsible for the content and writing the article. This study was supported by research grant VGST/P-8/CISE/2011-12/1151 from vision group on science and technology, Department of IT, BT, Science & technology, Govt. of Karnataka, Bangalore, Karnataka, India.

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