Studies On The Quality Control Of Herba Euphorbiae Humifusae And Pharmacokinetics Of Gallic Acid

Herba Euphorbiae Humifusae, herb of Euphorbia humifusa Willd. or Euphorbia maculata L., is an important crude drug with the effect of detoxicating and cooling blood to stop bleeding. In this dissertation, the chemical constituents, methods of quality control of Herba Euphorbiae Humifusae, and pharmacokinetic of active constituent gallic acid were studied in detail. Main contents were as follows:The chemical constituents of Herba Euphorbiae Humifusae were studied. 8 compounds were isolated by a combination of silica gel, polyamide and Sephadex LH-20 column chromatography. Their structures were identified as quercetin （1）, brevofolin carboxylic acid （2）, luteolin （3）, gallic acid （4）, luteolin-7-O-β-D-glucoside （5）, astragalin-6″-O-gallate （6）, quercetin-3-O-galactosyl-6″-O-gallate （7） and quercetin-3-O-β-D-glucoside （8）. Of the total, 2 and 3 were isolated from Herba Euphorbiae Humifusae for the first time.An HPLC quantitative analysis of gallic acid in Herba Euphorbiae Humifusae wasperformed on a Phenomenex C₁₈ column （250 mm×4.6 mm, 4 urn） with the mobile phase ofmethanol-0.5 % glacial acetic acid （10:90, v/v） and detective wavelength of 272 nm. Thelinear range of gallic acid was 1.75-35.0μg·ml^-1（r = 0.9993） and the recovery was 99.9 %（RSD = 1.8 %）. An HPLC method was established for the simultaneous determination of fiveflavonoids, namely quercetin-3-O-galactosyl-6″-O-gallate, luteolin-7-O-β-D-glucoside, quercetin-3-O-β-D-glucoside,apigenin-7-O-β-D-glucoside and kaempferol-3-O-β-D-glucoside.The separation was performed on a Phenomenex C₁₈ column （250 mm×4.6 mm, 4 urn） withthe mobile phase of methanol and water contained 0.1 % phosphoric acid （42.5:57.5, v/v） at aflow rate of 0.9 ml·min^-1 and detected at 330 nm. The linear ranges were 7.60-304μg·ml^-1 （r= 1） , 1.04-41.6μg·ml^-1（r = 0.9999） , 12.6-504μg·ml^-1 （r=.0.9999） , 0.82-32.8 ng·ml^-1（r = 0.9999） and 2.4-96.0μg·ml^-1 （r = 0.9999） , respectively. The recoveries of fiveflavonoids were 97.4 % （RSD = 2.4 %） , 98.0 % （RSD = 2.8 %） , 100.7 % （RSD = 2.8 %） , 98.1% （RSD=1.7%） and 101.7% （RSD = 2.8%）.A method was developed for the quantitative determination of gallic acid in rat plasma by high performance liquid chromatography （HPLC）, and then the pharmacokinetics in rats were studied. Acetaminophen was selected as internal standard, and the plasma samples were pretreated by protein precipitation with methanol. Phenomenex C₁₈ was used with the mobile phase of methanol and water contained 0.5 % glacial acetic acid （7:93, v/v） at a flow rate of 1.0 ml·min^-1, and detection wavelength was set at UV 272 nm. The linear range of gallic acid in rats plasma was 0.30-30.0μg·ml^-1（r = 0.9964）. Intra-day RSD and inter-day RSD were both no more than 11.7 %. The extraction recoveries of gallic acid exceeded 80.0 %. Then the method was succesfully applied to the pharmacokinetic study after intragastric （150 mg/kg） and intravenous （75 mg/kg） administration. After intragastric administration of gallic acid, t_1/2α, t_1/2β, T_max, C_max and AUC_0-t was 46.6 min, 56.5 min, 66.0min, 3.96 mg·L^-1and 396.5 mg·min·L^-1, respectively. As for intravenous injection, t_1/2α, t_1/2β and AUC_0-t was 9.9 min, 78.9 min and 461.9 mg·min·L^-1, respectively. After intragastric and intravenous administration, gallic acid showed to fit two-compartment model. Absolute bioavailability of gallic acid was 42.9 %.The equilibrium dialysis combined with HPLC to determine the plasma protein binding rate of gallic acid was carried out. Results showed that the plasma protein binding rate of gallic acid at low, middle, high concentrations were 81.4 %, 91.4 % and 83.2 %, respectively.