Study On Pharmacokinetics And Pharmacodynamics Of Effective Components From Herba Euphorbiae Maculatae

Euphorbi humifusa is attributed to Euphorbi, perennial herbage, having functions of detoxification hemostaasia, resisting pathogenic microbe, and so on. Resent researches indicate that Euphorbi humifusa mainly contents hydrolyzable tannic quercetin and flavonoids; Pharmacological researches prove it has many biological activitivies, such as bacteriostasis. oxidation resistance , liver protection hemostaasia. In summar, as a kind of low-costly and abundantly reproducible resource, effective components of Euphorbi humifusa have widely outlook for application and market. However, as far as studies about effective components and officinal of Euphorbi humifusa is concent, the in-depth research on function and mechanism is few now, and there is no pharmacokinetic and pharmacodynamic study about its extract in vivo of fish.The study optimized the extraction process of effective components of Herba Euphorbiae maculatae, extracted and separated total effective components of the herba , studied curative effects of the total effective components and gallic acid to the crucian bacterial septicemia and their pharmacokinetic courses in vivo, and researched the antimicrobial action and security of Herba Euphorbiae maculatae.1. Adopted stepwise comparison and the orthogonal experimental design, the extraction process of total flavonoids from Herba Euphorbiae maculatae was optimized, compared the influence of two solutions and two extraction methods on the content of total flavonoids of the extract, elicited the best method was continuous reflux method with ethanol, by which way the content of total flavonoids was up to 10.455 mg/g . Using orthogonal continous reflux method and orthogonal table L₉(3⁴), we investigated the effect of concentration of ethanol extraction times time and amount of solvent to the content of total flavonoids. The results indicated that the order of factors to the content was extraction times> time > amount of solvent > concentration of ethanol. According to analysis of variance, the optimum extraction process was 65% ethanol of 12 times quantity for medicine, extraction for 4 times and 2 hours for each time.2. The main components of total Herba Euphorbiae maculatae extract were analyzed by means of thin-layer chromatography (TLC). With a thin-layer silica GF254 plate and a mobile phase ofpetroleum ether-ethyl acetate-formic acid (50:50:0.6), observing the results of chromatography under UV light (254nm) or visible light under ammonia gas , we concluded that total Herba Euphorbiae maculatae extract contented many effective components such as quercetin, kaempferol and gallic acid. By using thin-layer chromatography and column chromatography, through silica gel column chromatography , with petroleum ether -ethyl acetate - formic acid (50:50:0.6) as separation cleanser , a kind of monomer was isolated, and it was proved to be gallic acid by its physical and chemical character.3. The antimicrobial action was experimented with Aeromonas sobvia as the representative pathogen offish bacterial septicemia, he results showed the minimum inhibitory concentration (MIC) of total effective components of Herba Euphorbiae maculatae against Aeromonas sobvia was 1.25mg/ml, the minimum bactericidal concentration (MBC) was 10mg/ml; gallic acid had a better inhibiting action to Aeromonas sobvia, its MIC and MBC were the same 3mg/ml. Give fish 1200mg/ml total Herba Euphorbiae maculatae extract and 96mg/ml gallic acid at a dose of 0.5ml/50g, there was no any ill responses within one week, and LD⁵0 had not been examined, indicating it was absolutely innocuous of total effective components of Herba Euphorbiae maculatae and gallic acid to the fish.4. The curative effects of total Herba Euphorbiae maculataeextract and gallic acid to crucian bacterial septicemia were studied by giving medicine through mouth. The results showed there was significant difference between cure groups and control group (P<0.05), while the difference between two cure groups was not significant (p>0.05), indicating their curative effects were obvious. Changes of biochemical indices indicated that total Herba Euphorbiae maculatae extract and gallic acid could obviously improve the ill condition of liver and kidney.5. The reverse-phase high-performance liquid chromatograph (HPLC) methods were developed to determine gallic acid in crucian's blood plasma. RP-HPLC analysis was performed by Waters-515 and the column was Kromasil C18. The HPLC conditions for analyzing gallic acid concentration in serum were as follows: the mobile phase was carbinol - 0.05mol/L phosphoric acid -water (4:16:50) ; the flow rate was 0.5ml/min ; the column temperature was room temperature ; pH of the mobile phase was regulated to 5.7 by triethylamine , sample size was 20ul , the UV detective wavelength was 271nm . Under above conditions , the average recovery of gallic acid in crucian's blood plasma was 100.1±4.76%, RSDof intra-day and inter-day were 2.89%å’ŒP 3.38% respectively .On the base of above analysis methods, the pharmacokinetics was studied after a single dose of...