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With an aging world population, osteoarthritis (Osteoarthritis, OA) rising prevalence, sick people is increasing. OA can cause pain, disability, seriously affect the patient's ability to work. OA occurs when the first damage the articular cartilage. Articular cartilage thinning, degeneration, destruction, subchondral bone sclerosis, joint space narrowing. Currently used for the treatment of OA of non-steroidal anti-inflammatory drugs and polysaccharides cartilage protective agent. Polysaccharides cartilage protective agents are generally heparin analogues, such as multi-sulfate glycosaminoglycan, multi pentosan sulfate, sodium sulfate glycosaminoglycan many other. Their role is to maintain the articular cartilage proteoglycan content, stimulate cartilage cells to synthesize collagen type Ⅱ and proteoglycan, which helps to maintain the integrity of the articular cartilage. Chondroitin sulfate (Chondroitin sulfate, CS) is an important component of joint cartilage, is a repeating disaccharide → 4)-β-D-GlcUA-(1 → 3)-β-D-GalNAc-(1 → glucosamine composition glycans. OA occurs when the cartilage damage, CS content decreased. supplement CS can effectively relieve symptoms, reduce inflammation, swelling of the region, down matrix metalloproteinase-9, reduce OA cartilage damage; may also reduce the incidence of joint temperature, pain clinical CS already on the combined oral medications or dietary supplements for osteoarthritis treatment and care of patients, while in-depth study, CS sulfate groups on the sugar chain with a variety of different locations isomers currently used in medicine multi CS extracted from animal cartilage tissue, which more ingredients of 4 - chondroitin sulfate (CSA), and 6 - chondroitin sulfate (CSC). while in human articular cartilage is mainly containing 4,6 - Chondroitin sulfate (CSE). vitro experiments found that when the analog OA occurs, cultured cartilage 4,6 - bis chondroitin sulfate content decreased, and targeted supplementary 4,6 - bis chondroitin sulfate at low concentrations to stimulate cartilage matrix synthesis and inhibition of protease synthesis, a significant protective effect of cartilage. tips sulfate and its negative charge may pathogenesis and treatment of OA are very important for the development of specificity for the pathogenesis of OA cartilage protecting agent The subject of the use of chlorosulfonic acid - formamide sulfonation of four - multi-sulfated chondroitin sulfate derivatives were prepared containing 4,6 - double chondroitin sulfate structure more sulfated chondroitin sulfate (Polysulfated chondroitin sulfate, PSCS ), the chondroitin sulfate to bring more negative charge, to change its conformation is the PSCS macromolecules with a large negative charge, easily absorbed orally, to develop a drug for oral treatment of OA is required to prepare a negatively charged less easily absorbed oral preparation. PSCS so we investigated the preparation, identification, formulation, and the pharmacological activity of rabbit knee OA. 1 PSCS Preparation, Purification and Characterization of chlorosulfonic acid - the CS-carboxamide multi sulphonation sulfation, preparation PSCS. Through SO 4 2 - Determination and SO 4 2 - / COO - ratio measurement results validate sulfonated, SO 4 2 - content increased from 12.76% to 20.62%, an increase of 7.76%; SO 4 2 - / COO - ratio rose to 3.35 from 1.35. yield was 78.9%. right product PSCS for capillary electrophoresis analysis and HPLC analysis, appears only a symmetrical peak, proved relatively pure product obtained has determined its physicochemical properties, including uronic acid content, hexosamine content, absolute molecular weight, specific rotation, etc. also measured PSCS anticoagulant potency of 40usp / mg. Description PSCS has some anticoagulant activity. 2 PSCS structure research UV, IR, 1 H-NMR and 13 C-NMR analysis of the structure of the PSCS . UV showed the sample does not contain conjugated double bonds, do not contain impurities such as proteins and nucleic acids. by comparison with CS IR confirmed the basic mother PSCS a CS-chain structure, which contains glucuronic acid and D-galactosamine disaccharide units , the β pyranose configuration. PSCS The IR and in the acid radical appeared in the galactosamine 6 peaks characteristic of carbon 818cm -1 , can determine galactosamine 6 -carbon on the acid-substituted hydroxyl After 1 H-NMR and 13 C-NMR analysis further, glucuronic acid, C-2, C-3 bits are connected on sulfate. PSCS disaccharide structure should mainly by 2,3 sulfated glucuronic acid and 4,6-position O sulfated galactosamine composition → 4)-β-D-GlcUA (2S, 3S) - (1 → 3)-β-D-GalNAc (4S, 6S) - (1 →. 3 PSCS screened for preparing oral preparation PSCS-chitosan nanoparticles formulations and methods of preparation conditions, the optimized conditions: shell The molecular weight of polysaccharide 5kDa, a concentration of 2mg ╱ ml, the solution pH6.0, added PSCS concentration 1mg/ml, a quarter of the volume of the chitosan solution dropwise at a rate of 2 sec / drop, drop overtime chitosan reaction mixture vortex speed 600r/min. proceeds PSCS-chitosan particle size of about 200nm, electric charge is 46mV around with good homogeneity, roundness and stability. 4 PSCS rabbit knee osteoarthritis cartilage protective effect with rabbit knee articular injection of collagenase Ⅱ established early models of osteoarthritis. daily gavage to PSCS-chitosan nanoparticles, sustained 4w. behavior, image , pathology testing confirmed the oral PSCS-chitosan nanoparticles on rabbit knee osteoarthritis have a certain effect. behavioral observation visible oral administration PSCS, the pain of osteoarthritis of the knee have a certain role in mitigation. X-ray film shows that the model group articular surface roughness, joint space narrowing, osteophytes suspected while smooth articular surface treatment group, only a slight narrowing of the joint space, no osteophytes. stripped cartilage, cartilage surface was observed: articular cartilage surface treatment group Compared with model group, malacia less, better gloss. observed by HE staining of cartilage biopsy, showing PSCS drug group can effectively protect the integrity of the cartilage surface and deep cells and structures; through Markin score, the results show that the model group 5.76 ± 3.23, PSCS low-dose group was 3.98 ± 1.88, PSCS high dose group was 3.32 ± 2.02. PSCS high and low dose group compared with model group were statistically significant, indicating that low-dose PSCS has a certain ease degenerative cartilage The role of sex change can be considered PSCS has some cartilage protection. detected in rabbit serum TNF-α and IL-1β levels. PSCS low dose TNF-α content was 8.98 ± 1.98pg/ml, with the model group 18.76 ± 1.23pg/ml compared with the significant difference, indicating PSCS can effectively inhibit osteoarthritis model in rabbit serum TNF-α is increased, and with the positive drug group, 9.92 ± 1.16pg/ml no significant difference in the effect. IL- 1β content PSCS compared with the model group was no significant difference in this issue the main achievements: ■ successfully fabricated high purity multi-sulfated chondroitin sulfate PSCS, and confirmed it contains 4,6 - double sulfate of cartilage hormone structure. ■ successful PSCS-chitosan nanoparticles were prepared and screened PSCS-optimal conditions for preparing chitosan nanoparticles. ■ The PSCS-chitosan nanoparticles for rabbit knee osteoarthritis treatment confirmed the PSCS early OA cartilage has obvious protective effect also verified PSCS-chitosan nanoparticles effectively absorbed by the body.
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