| Cow endometritis is a uterus mucosal inflammation caused by the infection of mucous membrane. The incidence rate is 20% to 50% in China. 60% to 90% of cow infertility is caused by endometritis, which is the main reason for the elimination of cows. Antibiotics were effective in treating cow endometritis, but drug residues, wasted milk and drug resistance are the main problems caused by using them. Suppositories were ease-of-use and have a high drug loading capacity, they can play a therapeutic role when entered the body and released between the fluid and vagina pressure. This study investigates the preparation, quality control and safety of the Lianhe suppository based on previous study, the aim is that providing a scientific basis for the new veterinary declaration and the clinical application of the suppository.1. The preparation of Lianhe suppository: The glycerides of mixed fatty acid were chosen as the suppository base, and the color and melt time of suppository was set as evaluation indexes, the preparation conditions of Lianhe suppository were optimized by orthogonal test, in addition, the displacement factor and the amount of theory quality of suppository were determined. The results showed that the optimum conditions was that the radio of solution of Lianhe and suppository was 1:1. The displacement factor is 1.38, and each Lianhe suppository(3 g) required 1.55 g of glycerides of mixed fatty acid.2. The antibacterial effects of Lianhe suppository in vitro: The antibacterial effects in vitro of Lianhe suppository were studied by agar diffusion plate method. The results showed that the bacteriostatic diameters of Lianhe suppository on Escherichia coli and Streptococcus were 15.39±0.67 mm and 19.79±0.68 mm respectively, which were considered highly sensitivity; to Staphylococcus aueus, the bacteriostatic diameter was 34.75±2.06 mm, which was considered extremely sensitivity. The data suggested that Lianhe suppository have a good antibacterial effect for the common pathogenic bacteria of cow endometritis.3. The Character and identification of Lianhe suppository: In this section, we investigated the physical properties and carried out the thin layer chromatography(TLC) of Lianhe suppository. The results indicated that the suppository surface was smooth and with uniform colour and lustrous, average weight was 3.1 g, pH was 6.1-6.3, melt time was 22 min, deflection temperature was 36.8℃ and melt range was 38.2℃-39.3℃. The developing solvent was cyclohexane-ethyl acetate-isopropyl alcohol-methanol-water-triethylamine(3:3.5:1:1.5:0.5:1). The results showed that Lianhe suppository with Coptidis rhizoma standard and berberine hydrochloride(BH) have the same spots in the same place under ultraviolet lamp(at 365 nm), and negative control without interference. TLC tested by chloroform-ethyl acetate-formic acid(6:4:1), showed that Lianhe suppository with Chebulae fructus standard and gallic acid(GA) have the same spots in the same place, and negative control without interference.4. The determination of effective compounds in Lianhe suppository by HPLC: High performance liquid chromatography(HPLC) was used for determining the content of BH and GA in Lianhe suppository. The results showed that the mobile phase consisting of acetonitrile-0.05mol/L KH2PO4(50:50), absorption at 345 nm, flow rate 1.0 mL/min, column temperature 30℃, the sample volume 10 μL, the regression equation was Y=3.9×106X-82692(r=0.9998), the range of 0.041616-0.83232 μg BH content and peak area within the sample showed a good linear relationship. Mobile phase consisting of methanol-0.5% phosphoric acid(5:95), absorption at 273 nm, the regression equation was Y=6.9×106X-39068(r=1.0000), the range of 0.201376-2.01376 μg GA content and peak area within the sample showed a good linear relationship. This method was stable, accurate, well reproducible and highly specific, which could be used for quality control of Lianhe suppository.5. Safety evaluation of Lianhe suppository: Acute toxicity, vaginal mucosa and eye conjunctiva stimulation experiments were used to evaluate the safety of Lianhe suppository. The results showed that LD50 of Lianhe suppository on mice was 794.33 mg/kg by intraperitoneal injection, and confidence interval was 684.42-921.98 mg/kg. Therefore, Lianhe suppository is considered as the low toxicity substances. Tissue sections showed that there were only slight irritation to vaginal mucosa, no irritation to conjunctiva of rabbits and no hemolysis or cruor. |
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