OBJECTIVE:To optimize the ethanol extraction process of anti-atherosclerosis effective fraction of Lindley eupatorium. METHODS:Using the contents of hyperin,quercetin,kaempferol and jaceosidin as indexes,the effects of ethanol volume fraction,liquid-solid ratio,extraction time and extraction times on ethanol extraction technology were investigated. By multiple index scoring method,orthogonal test was designed to optimize extraction technology,and validation test was conducted. RESULTS:The optimal extraction technology was 70% ethanol as extraction solvent with solid-liquid ratio of 12 : 1,reflux extracting for 3 times,2 h each time. Results of 3 batches of validation tests showed that the contents of 4 marker components were in high level,being 5.305 3,1.560 1,1.986 5,6.703 6 mg/g in average(RSD<1.1%,n=3). Results of validation tests were stable and reliable,and were in good agreement with the results of orthogonal tests. CONCLUSIONS:The optimized ethanol reflux extraction technology of L. eupatorium is stable and reliable,and performs well. Energy consumption and cost are suitable for industrial production.The results provide technology basis for further purification and enrichment of anti-atherosclerosis effective components of L.eupatorium.

Objective To investigate the inhibiting effect of CGP77675 (CGP),a Src inhibitor,on epithelial-mesenchymal transition (EMT) of human retinal pigment epithelial (RPE) cells induced by transformation growth factor-β1 (TGF-β1).Methods Human RPE cell line (ARPE19 cells) was cultured in vitro and divided into control group,TGF-β1 group and TGF-β1 +CGP group.Corresponding agent was added into culture medium based on grouping.The morphology of the cells were examined under the optical microscope 3 days after culture.The expressions of EMT-related genes and proteins in the cells were detected by real-time quantitative PCR and Western blot,respectively,including fibronectin 1 (FN 1),and plasminogen activation inhibitor 1 (PAI1),and the expressions of zonula occludens protein 1 (ZO1) and cytoskeleton protein filamentous actin (F-actin) were detected by immunofluorescence staining.MTT assay was employed to evaluate the cell proliferation rate.The migration distance of the cells was measured by scratch test.Results The ARPE19 cells in the control group showed an epithelial-like morphology and F-actin and ZO-1 were expressed along cell membrane.In the TGF-β1 group,the cells appeared to be fibrous-like,and the fluorescence staining of F-actin was disordered and ZO-1 was discontinuous on the cell membrane.The cells in the TGF-β1 +CGP group remained to be an epithelial-like in shape with clear and complete expressions of F-actin and ZO-1.The relative expressions of FN1 mRNA and PAI1 mRNA in the cells were 0.211 ± 0.080 and 0.116±0.073,1.000±0.001 and 1.000±0.001,0.368±0.097 and 0.362±0.048 in the control group,TGF-β1 group and TGF-β1 +CGP groups,showing significant differences among the groups (F=33.14,82.92;both at P＜0.01),with the highest expressions ofFN1 mRNA and PAI1 mRNA in the TGF-β1 group (all at P＜0.05).The relative expressions of FN1 and PAI1 proteins were 0.166±0.055 and 0.327±0.066,1.000±0.001 and 1.000± 0.001,0.143 ± 0.030 and 0.260 ± 0.077 in the control group,TGF-β1 group and TGF-β1 + CGP group,with significant differences among three groups (F=181.90,48.85;both at P＜0.01),and the expressions FN1 and PAI1 proteins were significantly higher in the TGF-β1 than those in the control group and TGF-β1 +CGP group (all at P＜0.05).The cell proliferative rate in the TGF-β1+CGP group was (79.30±3.44) ％ and (54.80±7.39) ％ at the third day and seventh day after culture,which were significantly reduced in comparison with (99.50 ± 1.00)％ and (99.10±0.50)％ in the control group as well as (95.10±4.20)％ and (92.10±4.50)％ in the TGF-β1 group (all at P＜0.05).The migration distance was disappeared in the TGF-β1 group,and the scratch width was not obviously changed in the TGF-β1 +CGP group.Conclusions Src inhibitor can inhibit EMT process of ARPE19 cells induced by TGF-β1,indicating that Src signaling pathway may play a critical role in EMT of RPE cells.