| Objective: To determine the optimum conditions of extraction of rough glycosaminoglycan from Perna viridis and after preliminary purification research its anti-tumor activity and investigate anti-tumor mechanisms in the aspect of immunomodulatory effect.Methods: 1. The optimum conditions of extraction of glycosaminoglycan from Perna viridis were determined by quadratic regression orthogonal rotational combing design studying the effects of three independent variables: solid-liquid ratio, enzyme dosage and enzymolysis time on the product of yield of crude glycosaminoglycan and content of glycosaminoglycan. 2. PVG-1 was obtained via freeze drying the treated crude glycosaminoglycan by dissolving the crude glycosaminoglycan and adjusting pH of solution to precipitate most of protein; the PVG-1 was purified by DEAE-52-cellulose chromatography. 3. The acute toxicity of PVG-1 was determined by the method of Test of Maximum Tolerated Dosage.4. MTT colorimetric assay was carried out to evaluate anti-tumor effect of PVG-1 and PVG-1a on human nasopharyngeal carcinoma cell CNE-2Z,chronic myelogenous leukemia cell K562 and osteosarcoma cell S180 in Vitro. Meanwhile the sensitization effect of PVG-1 and PVG-1a to 5-Fu were measured. On the basis of experiment in Vitro, the anti-tumor activity was verified via the experiment of inhabitation of PVG-1 on transplanted tumor S180 in Vivo. 5. The immune organs indexes were calculated to test the effects of PVG-1 on spleen and thymus index of normal mice,immunosuppressive mice and tumor-bearing mice; The effects of PVG,PVG-1,PVG-1a on phagocytic activity and activity of producing Nitro Oxide of mice peritoneal macrophages and on proliferation of mice spleen lymphocytes and on the cytotoxic activity of NK cells were studied in Vitro also.Results: 1. The optimum conditions of extraction of glycosaminoglycan from Perna viridis were acquired: solid-liquid ratio 2.30:1, enzyme dosage 0.94% and enzymolysis time 3.46h; Standard error of results of repeated verifing experiments and model prediction 25.50(×10-4)was 1.10%. 2. Content of glycosaminoglycan from Perna viridis was improved via preliminary refining PVG and one fraction PVG-1a was acquired through DEAE-52-cellulose chromatography. The content of glycosaminoglycan of PVG-1 and PVG-1a were 45.6% and 59.4% respectively. 3. Test of Maximum Tolerated Dose experiment showed that dose of PVG-1 >15.0 g·kg-1 in mouse acute oral toxicity test. 4. (1) PVG-1 exhibited inhibitory effect on K562 and CNE-2Z in Vitro. PVG-1 and PVG-1a showed obvious inhibitory effect on S180 in Vitro. PVG-1,PVG-1a could enhance anti-tumor effect of 5-Fu. (2) PVG-1 had obvious inhibitory effect on S180 in Vivo, with the inhibition rate of 60.6% of 200mg·kg-1 PVG-1. The inhibition rate of group of CTX together with middle dose of PVG-1 reached 78.8%. Middle dose of PVG-1 obviously improved the anti-tumor effect of CTX. 5. (1) PVG-1 could increase the spleen and thymus indexes of normal mice, and 200mg/kg,100 mg/kg PVG-1 could most obviously increase the spleen and thymus indexes of normal mice respectively. PVG-1 could repair low-immunity expression of immunosuppressive mice. (2) Each dosage groups of PVG-1 could increase the spleen and thymus indexes of tumor-bearing mice. Middle dose of PVG-1 (100 mg/kg) could repair low-immunity expression of tumor-bearing mice while treated by CTX. (3) PVG,PVG-1and PVG-1a could promote phagocytic activity and activity of producing Nitro Oxide of mice peritoneal macrophages and significantly promote proliferation of mice spleen lymphocytes in Vitro. (4) Each group of PVG,PVG-1and PVG-1a could promote cytotoxic activity of NK cells in Vitro. And the effect of groups of PVG-1a on cytotoxic activity of NK cells was better than PVG,PVG-1.Conclusion: The optimum conditions of extraction of GAG from Perna viridis were acquired. GAG from Perna viridis possessed the inhibitory effect on K562,CNE-2Z and S180, and improved the anti-tumor effect of 5-Fu and CTX. GAG from Perna viridis played a critical role in regulate immune function. One of the mechanisms of anti-tumor activity was it could regulate immune function. |
