Studies On Pharmacology And Clinical Application Of Flos Populi And It's Compound Preparation

Flos populi is the dry male inflorescence of Populus canadensis,populus tomentosa or same species plant. It's character is bitter-cold, and the pharmacological effect is clearing heat and detoxicate, dissipating dampness and zhili. But the system researches of Flos populi in pharmacology, pharmacodynamics, toxicology and the main active chemical composition are deficient. So it limits the exploitation in veterinary clinical. In this study, Flos populi compound injection composed of Flos populi and Scutellaria baicalensi was developed, and it was studied on preparation technology, pharmacology, toxicology, quality standards and clinical application. The results of research are summarized as following:1,Chemical composition of Flos populi aqueous extract,Ethyl acetate and n-butanol extract of Flos populi aqueous extract was tested by the method of test tube and thin-layer chromatography. The result showed that aqueous extract and it's Ethyl acetate extract contained polysaccharide, flavonoids, organic acid, quinones,lactones and coumarins, phenols and tanin, it may contain alkaloids, have no terpenoid and steroid, saponins, n-butanol extract contained flavonoids, actones and coumarins, organic acid and tanin.The content of flavonoids extracted were regarded as the assessing index, the extraction technology of Flos populi was studied by water-extraction and alcohol-precipitation and alcohol extracting method. The water-extraction and alcohol-precipitation technology was optimized by orthogonal design, the optimum extraction conditions are concluded as follow:Extraction solvent PH 7, extracted 2 times,2 hours for each time,65% alcohol precipitation,ultrafiltration solvent concentration less than 1.5g/mL, ultrafiltration temperature 15-45℃, ultrafiltration pressure exceed 0.05Mp.Salicin extarcted from Flos populi could be effctively purified by macroporous adsorption resin(MAR)AB-8 and silica gel column chromatography, In result, the purity of salicin after these steps was 24.17% and 96.23% respectively. The determination method of salicin was developed by HPLC on a C18 column, The methyl cuanide-water(7:93) as mobile phase. detection wavelength was 270 nm with column temperature 25℃and flowrate 1.OmL/min. The results showed that had a goodlinearity within the range 0.025μg-4.0μg (r=0.9994).2,The pharmacological effect of Flos populi extracts and the compound preparation were studied in the drug sensitivity test, the combined antibacterial activity test, the antibacterial infection test and anti-inflammatory effect test. The results showed that Flos populi aqueous extract and the compound preparation had moderate antibacterial activity against four tested bacteria (pathogenic and standard strains of E.coli and salmonella), Ethyl acetate and n-butanol extracts of Flos populi aqueous extract also showed a moderate antibacterial activity against pathogenic and standard strains of E.coli and standard strains of salmonella.In contrast, the antibacterial activity against the pathogenic salmonella was low, Flos populi and Scutellaria baicalensis had an additive effect on the antibacterial activity against E.coli.In the antibacterial infection and anti-inflammatory effect tests,the Flos populi aqueous extract and compound preparation had antibacterial activity against E.coli in vivo, and high,moderate and low dose groups of Flos populi aqueous extract can reduced the auricular swelling in mice, and the inhibition rates were 65.0%,58.9% and 51.6%, respectively,compared with cotrol group, the different was highly significant (P<0.01). High and moderate dose groups of Flos populi aqueous extract could significantly reduce the capillary permeability of mice, the inhibition rates were 57.02% and 53.84%, respectively, compared with control group, the difference was highly significant(P<0.01). The Flos populi aqueous extract could increase the immune organ indexes of mice. In conclution, the Flos populi aqueous extract and compound preparation possessed the significant antibacterial and anti-inflammatory effects.3,Acute toxicity and chronic toxicity of the Flos populi compound preparation were studied. In result, The LD50 Of the Flos populi compound preparation in mic ewas 46.4g/kg, and the 95% confidence interval was 46.40±10.22g/kg. The mice of the high,moderate and low dose groups were intraperitoneal injection administered for 28 days continuously with Flos populi compound preparation, The results show that these differences of indexes of blood rutine and biochemical, velocit of increase of weight, organ/body coefficients and histopathological examination were no significant compared with control group. The results showed that there is no obvious toxicity to mice after 28 days intraperitoneal injection administered, Indicating that it is safe to veterinary clinical administration.4,The preparation technology and stability of Flos populi compound injection were studied by the screening of auxiliary solvent, antioxidant, the investigation of the PH extent and roomtemperature retained sample observation test.In result, The expiration time of the Flos populi compound injection developed with auxiliary solvent(dimethyl-acetylamide),antioxidant(soduium or sodium hydrogen sulfite) was tentatively set on 1 years at 25℃.,the PH extent of injection is 6.5-7.5.The quality Standard of Flos populi compound injection was also studied in the test, establishing a method of HPLC to determinate the content of Baicalin in the injection. The results showed that these methods established was accurate and reliable for the quality control5,To study the eliminating effect and mechanism of drug resistance of Flos populi compound preparation against E.coli, the drug resistance E.coli isolated from clinical samples were detected by the drug sensitivity test and duplex PCR to detected the Quinolones and Sulfonamides-resistance genes (GyrA and Sul2), And the experiment of drug resistance elimination was performed in vitro with the drug resistance E.coli isolated from clinical samples as the target bacteria and Flos populi compound preparation as eliminating agent,Meanwhile the mechanism of the drug resistance elimination was detected by the analysis of the plasmid profile of E.coli, the sequence of gene (GyrA and Sul2) before and after eliminating action. Minimum Inhibitory Concentration (MIC) of Enrofloxacin and Sulfadiazine against E.coil was detected before and after eliminating action.The results showed that the E.coli isolated from clinical samples were high-level resistance, the drug resistance elimination rate of enrofloxacin,sulfadiazine-resistance and the control group was 19.5%,18.18% and 0, respectively. The growth rate of E.coli eliminated was step down. The counts of plasmid profile strap of E.coli and the genes (GyrA and Sul2) sequence were no change before and after eliminating test of drug resistant. In concluded, Flos populi compound preparation has the eliminating effect on the drug resistant of E.coli. The mechanism of eliminating action on enrofloxacin,sulfadiazine-resistance may not be the change in the drug resistance genes.6,To evaluate the clinical therapeutic effect of Flos populi compoud injection on swine infected with Escherichia coli artificially. The cure rate of Flos populi compoud injection of high,moderate and low dose groups were 90%,90%,80%, respectively. The cure rate of Audrographini control group was 60%.The results of clinical scale-up test showed that the total effective rate of Flos populi compoud injection was 97.14%, The cure rate was 84..28%, the Mortality was 2.86%.It was concluded that the clinical therapeutic effect Flos populi compoud injection was obvious.