| [Objective ] To explore the improved preparation of protoporphyrin disodium, observe the cytotoxicity of Napp in vitro and its inhibitory effect on HCV subgenomic replicon cell model.[Method] Protoheme was separated from protein hydrolysate of coagulated animal blood and protoporphyrin, protoporphyrin dimethyl ester and other intermediate products were extracted, which were finally made into protoporphyrin disodium. According to its light absorption characteristics, protoporphyrin disodium was quantitatively detected.Few cytotoxic concentration were measured by Cell Counting Kit-8.Add 10μl complete culture medium containing various concentrations of Napp or IFN to the plate of HCV subgenomic replicon cell.the top concentration of Napp and IFN were 10mg/ml, 106 IU/ml respectively. Every group was at a dilution of 1:10. After 72 hours cytotoxicity was measured by CCK-8 kit at 450nm aborsbance. Then the HCV subgenomic replicon cell model was incubated with Napp at concentration of 1mg/ml, 10-1mg/ml, 10-2mg/ml, 10-3mg/ml, 10-4mg/ml in cell culture solution and the negative and positive controls were set up correspondingly. After 3 days, the intracellular RNA of HCV subgenomic replicons were detected by FQ-RT PCR.[Result] The extract was henna crystal which was soluble in water and insoluable in chloroform, ether and acetone and it was easily soluble in acid solvent. The purity of the extract in acid solvent was about 93.8% by UV spectrophotometry. The range of few cytotoxic concentration of Napp varied from 0μg/ml to 1000μg/ml and at concentration from 10μg/ml to 1000μg/ml could significantly decrease the level of intracellular RNA of HCV subgenomic replicon cell model(P<0.050.01).[Conclusion] The protoporphyrin disodium can be extracted from inanticoagulant animal blood through the improved method and its purity is 93.8%. Napp has little cytotoxic effect on target cells and it can effectively inhibit the RNA level of HCV subgenomic replicon cell model.
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