| Cow endometritis is a harmful reproductive disease, which is caused by bacteria infection. Chinese traditional medicines have unique advantages for the treatment of cow endometritis. Danlianhua uterine perfusate is formed by Coptidis rhizoma, Forsythiae fructus, Salvia miltiorrhizae radix et rhizoma and Carthami flos through water decoction, has the function of antibacterial and anti-inflammation, and has a significant effect for the treatment of acute endometritis. Scientific and reasonable quality standard of Chinese medicine is the premise and foundation for clinical medication safety and validity. This experiment has carried on the qualitative, quantitative and stability of Danlianhua uterine perfusate. The results were as follows:(1) Identification of the main components in the Danlianhua uterine perfusateIdentification of berberine hydrochloride: The smple, the negative sample, the control herbs of Coptidis rhizoma, the herbs of Coptidis rhizoma, berberine hydrochloride, pointed the same high efficiency silica gel G plate with 1 μL, developed by cyclohexane-acetidinisopropyl alcohol-methanol-water-triethylamine(3:3.5:1:1.5:0.5:1) after saturated with ammonia, viewed under UV light of 365 nm. The chromatogram of the sample had 4 fluorescent spots of the same color in the corresponding position with the control herbs of Coptis rhizoma and had the same fluorescent spots with the berberine hydrochloride.Identification of phillyrin: The smple, the negative sample, the control herbs of Forsythia fructus, the herbs of Forsythia fructus, phillyrin, pointed the same silica gel G plate with 5 μL, developed by methane-methanol(3:1) after saturated with ammonia, and sprayed 10% ethanol sulfate solution for heating color. The chromatogram of the sample had the same color spots in the corresponding position with the control herbs of Forsythia fructus and phillyrin.Identification of salvianolic acid B: The smple, the negative sample, salvianolic acid B, pointed the same silica gel GF254 plate with 1 μL, the herbs of Salvia miltiorrhizae radix et rhizoma, pointed the same silica gel GF254 plate with 0.5 μL, developed by methylbenzeneacetidin-methane acid(5:6:1), viewed under UV light of 245 nm. The chromatogram of the sample had the same color spots in the corresponding position with the salvianolic acid B.Identification of hydroxysafflor yellow A: The smple, the negative sample, the control herbs of Carthami flos, the herbs of Carthami flos, hydroxysafflor yellow A, pointed the same silica gel G plate with 3 μL, developed by acetidin-methane acid-methane-water-methanol(7:2:3:0.4), viewed under white light and UV light of 365 nm. The chromatogram of the sample had the same color spots in the corresponding position with the control herbs of Carthami flos and hydroxysafflor yellow A.(2) Content determination of main components in the Danlianhua uterine perfusateContent determination of berberine hydrochloride: Chromatographic experiments were performed on Diamonsil C18(5 μm, 150×4.6 nm) with acetonitrile-0.05mol/L potassium dihydrogen phosphate(50:50) as the mobile phases. The column temperature was maintained at 30℃, the flow rate was 1m L/min, the detection wavelength was 345 nm, and 10 μL of samples were injected in every case. Regression equation was Y=38553X-89683, r=0.9998, which showed that berberine hydrochloride had a good liner relationship within the range of 4.1616-83.232 μg/m L.Content determination of phillyrin: Chromatographic experiments were performed on Diamonsil C18(5 μm, 150×4.6 nm) with acetonitrile-water(25:75) as the mobile phase. The column temperature was maintained at 30℃, the flow rate was 1m L/min, the detection wavelength was 277 nm, and 10 μL of samples were injected in every case. Regression equation was Y=7×106X+8636, r=0.9999, which showed that phillyrin had a good liner relationship within the range of 0.02287-0.3659 mg/m L.Content determination of salvianolic acid B: Chromatographic experiments were performed on Diamonsil C18(5 μm, 150×4.6 nm) with methanol-acetonitrile-methane acidwater(30:10:1:59) as the mobile phase. The column temperature was maintained at 30℃, the flow rate was 1m L/min, the detection wavelength was 286 nm, and 10 μL of samples were injected in every case. Regression equation was Y=1×107X-80303, r=0.9999, which showed that salvianolic acid B had a good liner relationship within the range of 0.02952-1.476 mg/m L.Content determination of hydroxysafflor yellow A: Chromatographic experiments were performed on Diamonsil C18(5 μm, 150×4.6 nm) with methanol-acetonitrile-0.7% phosphate(26:2:72) as the mobile phase. The column temperature was maintained at 30℃, the flow rate was 1m L/min, the detection wavelength was 403 nm, and 10 μL of samples were injected in every case. Regression equation was Y=3×107X-30671, r=0.9998, which showed that hydroxysafflor yellow A had a good liner relationship within the range of 0.020424-0.20424 mg/m L.Content determination of kaempferol: Chromatographic experiments were performed on Diamonsil C18(5μm, 150×4.6nm) with methanol-0.4% phosphate(52:48) as the mobile phase. The column temperature was maintained at 30℃, the flow rate was 1m L/min, the detection wavelength was 367 nm, and 10 μL of samples were injected in every case. Regression equation was Y=38387X-84646, r=0.9995, which showed that kaempferol had a good liner relationship within the range of 2.4232-96.928 μg/m L.Methodology verification experiment results showed that the above HPLC methods established were highly applicable, reproducible and stable, which could be used for the content determination of berberine hydrochloride, phillyrin, salvianolic acid B, hydroxysafflor yellow A and kaempferol of Danlianhua uterine perfusate.(3) Stability test of Danlianhua uterine perfusateDanlianhua uterine perfusate was placed for ten days in the environment of the illumination(4500 Lx±500 Lx), recorded the color, clarity and p H of the perfusate at the fifth day and the tenth day, identified the salvianolic acid B and hydroxysafflor yellow A and determined their contents. The results were that the color, clarity and p H of the perfusate had no obvious change, the contents of the hydroxysafflor yellow A decreased slightly and the contents of salvianolic acid B decreased significantly, which showed that the salvianolic acid B was sensitive to the light, and the preparation should be kept away from the light. |
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