| Objective: To perform an effective component analysis of Flos Lonicerae Japonicae.Methods: The HPLC method was used for determination the content of chlorogenic acid in Flos Lonicerae Japonicae. Chromatographic column was VP-ODS C18 column (150mm×4.6mm) and the mobile phase was acetonitrile-0.4% phosphonic acid (13:87), detection wavelength was 327nm. The UV-VIS was used to determine absorption of Flos Lonicerae Japonicae. solution on 490nm detection wavelength. With the rutin as comparison, the content of flavonoids was calculated; The HPLC method was used to determine the content of luteoloside in Flos Lonicerae Japonicae. A VP-ODS C18 as the column (150mm×4.6mm) and the methyl cyanide as mobile phase A, 0.5% glacial acetic acid solution as mobile phase B, 350nm as detection wavelength, the gradient elution was carried out in 0~30min according to A (%) 10→30, B (%) 90→70.Results: There was a good linear relationship between the quantities of the chlorogenic acid sample (0.2~1μg) and their peak areas (r=0.9996). The content of chlorogenic acid in Flos Lonicerae Japonicae. was 2.72%. There was a good linear relationship between the absorbance and the concentration of rutin within the range from 8~40μg/ml (r=0.9998). The content of flavonoids in Flos Lonicerae Japonicae. was 6.46%. The determination of luteoloside showed a good linear relationship in the range of 0.08～0.4μg (r=0.9995). The content of luteoloside in Flos Lonicerae Japonicae. was 0.065%.Conclusion: The chlorogenic acid and the flavonoids are both the main effective components of Flos Lonicerae Japonicae. Part 2:Studies of the extraction from Flos Lonicerae. inhibiting macrophagecell cholesterol cumulative actionObjective: To observate the extraction from Flos Lonicerae. inhibiting macrophage cell cholesterol cumulative action.Methods: Using the extraction and separation technology of the Chinese Traditional Drugs, the ethanol extracts from Flos Lonicerae were obtained. The ethanol extracts was extracted with petroleum ether, ethyl acetate, n-butanol according to the different polarity of solvent, the extracts of petroleum ether phase, ethyl acetate phase, n-butanol phase and water phase were obtained respectively. Petroleum ether phase, ethyl acetate phase and n-butanol phase as test drugs (drug group 1~3) were carried out the next experiment. Mouse macrophage cells (RAW264.7 cells)were cultivated in vitro, with the different concentrations of fat emulsion (20% fat emulsion and culture medium proportion respectively were 0:1,1:6,1:5,1:4,1:2,1:1) incubated the RAW264.7 cells, next with the best concentration fat emulsion incubated cells different time (0,3,6,12,24,48h), the Oil Red O staining observated fat granule situation of cells. In the further experiments, cells were divided into 5 groups. That were the normal group, 1ipid-loaded cell (10% fat emulsion) group, 10% fat emulsion+drug1 group, 10% fat emulsion+drug2 group, 10% fat emulsion+drug3 group. The Oil Red O staining observated fat granule changes in cells. Caveolin-1 expresses were detected by Western blotting and SREBP-1mRNA expresses were detected by the RT-PCR.Results: The Oil Red O staining results showed that the fat granule increased with fat emulsion concentration and incubating time increasing in the appropriate concentration and time range. It had the best effect when 10% fat emulsion incubated cells for 24h; After cells were treated by different factors for 24h, the Oil Red O staining results showed that drug1 group, 2 group, 3 group caused a fat granule of reduction in the cells, and the effect of the drug2 group was the most obvious. The Western blotting result showed that 1ipid-loaded cell group of caveolin-1 expression declined comparing with the normal group, the drug groups (drug group 1~3) of caveolin-1 expressions had the upward tendency comparing with the 1ipid-loaded cell group, and the durg2 group of upwards was the most obvious; Immunofluorescence detection showed that drug groups (drug group 1~3) caveolin-1 were up-regulated expressions comparing with the 1ipid-loaded cell group, and the drug2 group of up-regulation was the most obvious. The RT-PCR result showed that SREBP-1mRNA expression was up-regulated of the 1ipid-loaded cell group comparing with the normal group. SREBP-1mRNA expressions of the drug groups (drug group 1~3) were down-regulated comparing with the 1ipid-loaded cell group.Conclusion:1. Flos Lonicerae. extraction (drug1,2,3) can inhibit macrophage cell cholesterol accumulation, and the drug2 is most efficient than others;2. Flos Lonicerae. extraction inhibiting macrophage cell cholesterol accumulation is perhaps related to caveolin-1 expression up-regulated;3. Drugs can up-regulate caveolin-1 expression by inhibiting SREBP-1 expression;... |
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