| Yejuhua injection was studied in this dissertation. In order to isolate the extraction products of ethyl acetate from Flos Chrysanthemi Indici, various chromatographic techniques were performed. By NMR data and literature four compounds were isolated and identified. They were linarin, luteolin-7-glucoside, chlorogenic acid and caffeic acid.The anti-tumour activity in vitro of luteolin-7-glucoside isolated from Flos Chrysanthemi Indici was reported in this dissertation. The studies demonstrated that luteolin-7-glucoside inhibited the proliferation of human HepG2 cells in a time- and dose-dependent manner with a single dose of 40μg·mL-1. MTT assay, hotomicroscopical observation, fluorescence microscopical observation and DNA agarose gel electrophoresis showed that luteolin-7-glucoside induced cell apoptosis in HepG2 cells.RP-HPLC method was developed for the simultaneous determination of chlorogenic acid and caffeic acid in Flos Chrysanthemi Indici. The linear ranges for chlorogenic acid and caffeic acid were 1.0～20.0μg.mL-1 (r = 0.9997) and 0.053～1.1μg.mL-1 (r = 0.9998), respectively. The average recoveries were 97.9% (RSD = 2.2%) and 99.2% (RSD = 2.5%), respectively. In the meantime, the contents of chlorogenic acid and caffeic acid in Yejuhua injection were determined. The average recoveries were 101.4% (RSD = 1.5%) and 99.4% (RSD = 2.6%), respectively. RP-HPLC method was developed for the simultaneous determination of luteolin-7-glucoside and linarin in Flos Chrysanthemi Indici. The linear ranges were 0.2～4.2μg·mL-1 (r = 0.9998) for luteolin-7-glucoside and 2.1～41.2μg·mL-1 (r = 0.9998) for linarin. The average recoveries were 98.6% (RSD = 1.8%) and 99.4% (RSD = 1.7%), respectively. In the meantime, the contents of luteolin-7-glucoside and linarin in Yejuhua injection were determined. The average recoveries were 99.3% (RSD = 1.5%) and 99.9% (RSD = 1.3%), respectively. The essential oil in Flos Chrysanthemi Indici was analyzed by GC-MS. 56 compounds were got and 34 were identifed in the oil.A HPLC fingerprint was developed based on 17 batches of Flos Chrysanthemi Indici. There were 24 common peaks in the fingerprint and 5 were identified. A HPLC fingerprint was developed based on 13 batches of Yejuhua injection. There were 16 common peaks in the fingerprint and 4 were identified. Furthermore, by comparing the fingerprints of injection, Semi-manufactured product and Herb, the good correlation of peaks between fingerprints were found.RP-HPLC method was developed for the simultaneous determination of luteolin-7-glucoside and linarin in Beagle dog plasma. After being deproteinized by acetonitrile, the plasma samples were analyzed with rutin as internal standard. The assay was shown to be linear over the range of 3.9～1560 ng·mL-1 (r = 0.9928) for luteolin-7-glucoside and 3.4～1373 ng·mL-1 (r = 0.9941) for linarin. Mean recoveries were 82.6% and 74.2%, respectively. The HPLC method developed has been applied to determine the pharmacokinetics of luteolin-7-glucoside and linarin in Beagle dogs plasma after having taken Yejuhua injection via intramuscular route. The pharmacokinetic parameters were calculated. For luteolin-7-glucoside (99.3μg/kg), the time for peak plasma level (Tmax) was 0.37 h and the peak plasma level (Cmax) was 61.75 ng/ml. The area under concentration-time curve (AUC0-∞) was 79.30 ng·h·mL-1. The elimination rate constant (Ke) and the biological half-life (T1/2) was 1.02 h-1 and 1.12 h, respectively. While those parameters for linarin (173.2μg/kg) were 0.42 h, 134.9 ng/ml, 188.9 ng·h·mL-1, 0.67 h-1 and 1.08 h, respectively.The method for the determination of linarin in Beagle dogs plasma has been optimized. The plasma samples were analyzed after being deproteinized by acetonitrile and daidzein was used as an internal standard. The assay was shown to be linear over the range of 6.4～2575 ng·mL-1 (r = 0.9966). Mean recovery was 77.0%. The method was used to determine linarin in Beagle dogs plasma after intramuscular administration of linarin solution at a dose of 177.7μg/kg. The pharmacokinetic parameters were as follows: the Tmax and Cmax were 0.70 h and 37.87 ng/ml, respectively. The AUC0-∞ was 65.74 ng·h·mL-1. The Ke and T1/2 were 0.51 h-1 and 1.44 h, respectively. After intravenous administration of luteolin-7-glucoside solution to Beagle dogs at a dose of 95.5μg/kg, the pharmacokinetic parameters were as follows: AUC0-∞, 22.61 ng·h·mL-1; Ke, 1.66 h-1 and T1/2, 0.48 h, respectively.RP-HPLC method was developed for the simultaneous determination of luteolin-7-glucoside and linarin in rat plasma. After being deproteinized by acetonitrile, the plasma samples were analyzed with rutin as internal standard. The assay was shown to be linear over the range of 22.8～4560 ng.mL-1 (r = 0.9969) for luteolin-7-glucoside and 20.8～4160 ng·mL-1 (r = 0.9970) for linarin. Mean recoveries were 70.7% and 66.6%, respectively. The method was used to determine luteolin-7-glucoside and linarin in rats plasma after intramuscular administration Yejuhua injection. For luteolin-7-glucoside (0.271μg/g) , the pharmacokinetic parameters were: Tmax, 0.14 h; Cmax,98.8 ng·mL-1; AUC0-∞,194.0 ng·h·mL-1; Ke,0.43 h-1 and T1/2,1.74 h, respectively. While those parameters for linarin (0.632μg/g) were 0.13 h, 323.5 ng/ml, 533.1ng·h·mL-1,0.57 h-1 and 1.27 h, respectively.The method for the determination of linarin in rat plasma has been optimized. The rat plasma samples were analyzed with daidzein as internal standard after being deproteinized by acetonitrile. The assay was shown to be linear over the range of 20.8～4160 ng·mL-1 (r = 0.9935). Mean recovery was 67.7%. The method was used to determine linarin in rats plasma after intramuscular administration of linarin solution at a dose of 0.713μg/g, the pharmacokinetic parameters in rats plasma were as follows: Tmax, 0.30 h; Cmax, 394.1 ng·mL-1; AUC0-∞, 683.8 ng·h·mL-1; Ke, 0.86 h-1 and T1/2, 1.09 h, respectively. After intravenous administration of luteolin-7-glucoside with a dose of 0.297μg/g, the pharmacokinetic parameters in rats plasma were as follows: AUC0-∞, 79.21 ng·h·mL-1; Ke, 1.82 h-1 andT1/2, 0.54 h, respectively.The studies demonstrated that the absorption of luteolin-7-glucoside and linarin in Beagle dog and rat after administration of Yejuhua injection were better than that of single chemical. Furthermore, the administration of Yejuhua injection resulted in a longer T1/2, increased the residence time for luteolin-7-glucoside and improved the efficacy of medicine than those after administration of single chemical. The studies indicated that it is the co-operated effects among the chemical components of traditional Chinese medicine that made the administration of Yejuhua injection more effective than that of single chemical from the point of pharmacokinetics. |
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