Humaira Majeed Khan

Isolation, Characterization Of Chondroitin Sulphate And Its Efficacy In Osteoarthritis - 2012

Chondroitin sulphate (CS) and Glucosamine sulphate (GS) are two main components of articular cartilage. It is believed that these molecules slow down wear and tear of cartilage. Moreover, if administered exogenously as drugs, these may initiate synthesizing capacity of cartilage. Among these, GS promotes the formation and repair of cartilage, whereas CS promotes elasticity and prevent cartilage breakdown by inhibiting degradative enzymes. Concurrent use of both structural units of cartilage as drugs in osteoarthritis (OA) may lessen the progression of disease.
The present study was conducted to elucidate the chicken keel cartilage as an alternate and potential source for this endogenous component that may be used exogenously to repair or prevent damage to joints. Chicken keel cartilages were collected from healthy broilers. CS was extracted using MgCl2 solution (3M), dialyzed and digested with papain. The extracted material was purified by ethanol precipitation, centrifugation and then freeze dried. Proximate analysis of semi-purified polysaccharides revealed the presence of carbohydrates (65.49±0.10), crude protein (12.82±0.26), ash (11.12±.56), moisture (9.88±0.32) and fat (0.69±0.14). Fiber contents were found to be nil in the processed samples. Dimethylmethylene blue binding (DMMB) assay was performed for determination of percent contents of CS in extracted semi-purified samples and mean concentration was found to be 70.77±2.35. Semi-purified polysaccharides were further characterized by FTIR (Fourier Transform Infrared Spectrometer) technique and characteristic Peaks of CS molecules were recorded at 854, 854 and 853 cm-1 and then compared with spectrum of standard CS. Protein content being a major impurity in extracted samples was determined by Bradford method quantitatively (4.64±0.29). Two protein impurities having 77.8 and 50.5 kDa molecular weights were revealed by SDS-PAGE.
Efficacy of semi-purified CS from chicken keel cartilage, standard CS from shark source and GS, alone and in combination in experimental OA rat model was evaluated. To develop OA similar to spontaneous OA, 10mg papain/0.5mL (Sigma, Cat # P 3125) in buffered solution of 0.05 M sodium acetate pH 4.5 was injected intra-articularly in each right knee joint of fifty five albino rats (pre-anesthetized with anesthetic ether). Ten rats (n= 10) were injected with 0.5mL of normal saline (0.9%) in right knee joint that served as control group. Then from fifty five papain injected rats, twenty five were divided into five groups (n=5) for development and assessment of OA model (OA groups). Progression of disease was monitored by clinical scores, histopathological scores and concentration of CTX-II as biomarker in sera samples of experimental rats by ELISA using a commercial kit (serum preclinical CartiLaps ® ELISA kit) for control and OA groups (n=5) on day 0 (control group) and days 1st, 7th, 14th, 21st and 28th post papain injection (OA groups). Highest mean clinical score (10.38±1.1) was observed on 1st day and least on 28th day post papain injection i.e. 5.00±.34. Highest mean histopathological score and CTX-II concentration was recorded on 28th day i.e. 12.82±1.64 and 36.82±3.81. Values of clinical scores, histopathological scores and CTX-II concentration reached to maximum on 21st day and then sustained thereon. Second phase of experiment is comprised of evaluating and comparing the efficacy of extracted CS samples (chicken keel cartilages), standard CS (shark source) alone and in combination with GS. For this purpose, remaining five rats out of ten injected with normal saline intra-articularly served as control groups along with treated and non treated groups of experimental rats. Remaining thirty OA induced rats were divided into six groups (five rats /group). Group 1 (n=5) called non treated group received only placebo till 60th day and served as negative control group.
Treated Group 2 received GS alone, Group 3 CS (standard) and Group 4 were given extracted CS. Group 5 was treated with combination of GS plus CS (standard) and Group 6 with GS plus CS (sample). Doses of glycosaminoglycans (GAGs) were administered as 1.2g/kg/day CS and 1.5g/kg/day GS alone and in combinations. Drugs were offered early in the morning in bolus form with feed (10g) after overnight fasting while non-treated group received only placebo (without any drug).
Anti-arthritis activities of CS standard and extracted alone and in combination with GS were assessed clinically, analyzed statistically by using one way ANOVA. Level of significance (P<0.05) was recorded by using Duncan's Multiple Range (DMR) Post hoc Test. Mean scores of clinical, histopathology and CTX-II concentrations observed at 60th day in control rats (without OA) were 0.00, 0.00 and 2.55, respectively. OA induced untreated group showed mean score for clinical signs, histopathological scores and CTX-II concentrations 4.15, 12.24 and 36.70 and GS treated group 3.19, 3.96 and 6.12 at 60th day of treatment, respectively. For CS (standard), mean scores of clinical signs, histopathological lesions and CTX-II concentrations were recorded as 2.64, 2.44 and 4.48 and for CS (extracted) were 2.26, 2.28 and 4.40 in sera correspondingly at 60th day of treatment. The lowest mean values of clinical signs, histopathology and CTX-II concentrations in sera of treated group with standard CS plus GS were found to be 0.94, 0.94 and 2.62 followed by extracted CS plus GS treated groups 01.05, 1.27 and 2.74, respectively. Clinical, histopathological scores and CTX-II concentrations in group of rats treated with combinations were found to reverse the diseased condition after 60th days of treatment as the values were close to that of normal rats and far away from OA rats. It is concluded that extracted CS from poultry has comparable efficacy with CS standard from shark source alone and in combination with GS. Poultry by-product (keel cartilage) is found to be an alternate and cheap source for CS (chondroprotective agent) as compare to expensive, less available and religiously prohibited source for Islamic countries particularly.

Department of Pharmaoclogy & Toxicology
Phd. thesis


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