Preparation Of Chaenomelis Fructus Polysaccharides And Anti-rheumatoid Arthritis Effect

Chaenomeles speciosa is a genus of Chaenomeles which belongs to the family of Rosaceae. Its dry fruit ---Chaenomelis Fructus, also called Zhou Pi Mu gua or Tie Geng Hai Tang in Chinese. It has been used for many years. Chaenomeles speciosa has been included in many herbal formulations, which was used as a harmonize formula in Traditional Chinese Medicine (TCM). The first written reference to the species dates as far as 401 B.C. Its fruit has sour and warm properties, and it could regulate meridians (Ching Lo, Classical loci in acupuncture) of Liver and Spleen. In addition, it could activate the flow of Qi and blood, and to regulate the stomach. What is more, it contains a variety of chemicals, including phenols, lignans, triterpenes, flavones, minerals, vitamins, amino acid, sterides, protein and other constituents. Until now, a great many of pharmacological reseach was carried out about Chaenomeles speciosa. Polysaccharides are a hotspot in recent years since they possess a variety of pharmacological activities. However, there is no scientific report available in the literature on the anti-inflammatory effect and anti-rheumatoid arthritis effect of Chaenomelis Fructus polysaccharides(CFP). Therefore, this paper mainly study the anti-rheumatoid arthritis activity of CFP in vitro and in vivo. Specific details of study are as follows:Part Ⅰ Preparation of CFPThe content of total carbohydrate was determined by the phenol-sulfuric acid colorimetric assays using glucose as the standard at 490.0 nm. Variability test, stability test, repeatability test and sample recovery rate test are fit the bill. The best hot water extraction process and ethanol precipitation from Chaenomelis Fructus polysaccharides was carried out by L9(34) orthogonal experiment. Trichoroacetic acid method was selected to remove protein. At final, the purity of CFP reached 52.97%.Part Ⅱ Effect of CFP on inflammation originated from LPS in vitroThe toxicity of CFP was tested by cell counting kit to search for safety and effective dosages. CFP did not significantly exhibit cytotoxicity at the range of 0～200 mg/L against NR8383 cells. This dose range was used for treatment of CFP in the following experiments. The treatment with CFP considerably inhibited the production of TNF-a, IL-1β, COX-2 and NO and accosiated with the doses in a negative manner. CFP was assigned to anti-inflammatory potentiality in great degree.Part Ⅲ Anti-rheumatoid arthritis effect of CFP on animalsThe administration of CFP (12.5,25,50 mg/kg body weight) downregulated the volume value of joint swelling induced by CFA in a dose dependent. The maximum inhibition rates were 21.38%,31.03% and 33.10%, respectively. Injections of CFA in the right hind paw also induced secondary chronic arthritis. The CFP inhibited the secondary arthritis of the left hind paw on a dose-dependent among the dose of 12.5-50 mg/kg and with the maximum inhibition rate of 24.39%,41.46% and 51.11%, respectively. Histopathological assessment of paraffin embedded sagittal sections of ankle joints with H&E stain indicated that administration of the CFP reduced cellular infiltration in the CFA-induced rats. The pannus was partly inhibited. The damage of articular cartilage was relieved. In addition, CFP promoted a marked decrease the infiltration of cells. Treatment of different doses of CFP and standard drugs did not shown any weight loss. Thus CFP had no toxicity.No evident effects were observed. It was demonstrated that CFP at a dose of 12.5,25 and 50 mg/kg have a dose-dependent inhibition tendency of paw edema volume induced by carrageenin.CFP showed a significant analgesic effect against acetic acid-induced writhing response in mice(P<0.01) in a dose dependent manner. The inhibition percentage at each dose of CFP (25 mg/kg,50 mg/kg and 100 mg/kg) were 11.40%,22.79% and 49.57%, respectively. The inhibition rate ranked as 100 mg/kg CFP>Aspirin>50 mg/kg CFP>25 mg/kg CFP.CFP decreased HAc-induced vascular permeability in comparison to the control group. A dose-dependent inhibitory activity was observed at the dose of 25-100 mg/kg. The highest inhibitory value was 55.97% at the dose of 100 mg/kg. The lowest inhibition rate was 26.75% at the dose of 200 mg/kg.In the experiment of Cy-induced myelosuppression, mice with CFP treatment showed remarkable decrease in levels of phagocytic index. The phagocytic index of the CFP was lower than that of the control group at the dose of 25 mg/kg and 50 mg/kg(P<0.05). In addition, CFP significantly suppressed macrophage phagocytic activity measured by the phagocytic index at the dose of 100 mg/kg. It decreased the carbon clearance but no significant as comparing to Cy group. The phagocytic index for CFP was lower comparing to control group.There was dose-dependent decrease in haemagglutinin titre in immunosuppressed animals indicating that the CFP is effective to arthritis by down-regulating humoral immunity. Administration of CFP resumed in decreased haemagglutinin titre. The result was significant(P<0.01, P<0.05) when compared to control animals at the doses of 100mg/kg and 50 mg/kg.Part IV Isolation and purification of CFPCFP was purified through DEAE-650 M and washed with aqueous sodium chloride(0mol/L,0.1 mol/L, 0.2 mol/L)in turn. Thus CFP made up of three parts. experiment found that 0 mol/L and 0.1 mol/L NaCl fractions showed anti-inflammatory activity while0.2 moI/L NaCl fraction had no anti-inflammatory activity. 0 mol/L and 0.1 mol/L NaCl fractions were puried by Sephacryl S series are still under way.In summary, we have demonstrated that CFP inhibhed 1;he expression of pro-inflammatory mediators such as TNF-a and IL-ip and displayed anti-inflammatory ect in v/Yra(LPS-induced NR8383 macrophage cells). CFP has notable anti-rheumatoid arthritis effect and analgesic activity. The results provide a scientific basis for folk medicine for t;he treatment of inflammation. The conclusions also suggests that CFP has a good prospects to treat rheumatoid arthritis.