| Caryophylli Flos(CF) is the dried bud of Myrtle familyplant of Eugenia caryophyllata Thunb. CF is spicy-warm, with functions such as warming middle burner and antiemetic, tonify the kidney yang, mainly used for treatment of syndromes like abdominal fullness and distention, diarrhea and kidney deficiency, and so on. Study of chemical composition has shown that CF mainly contains volatile oil(17.1%), in which eugenol content up to 80.3%. Pharmacological studies have shown that CF and components have significant anti-bacterial, anti-mites and anti-oxidant activities. The advantages of microemulsion are thermo dynamically stable, low viscosity, good dispersibility, and conducive to absorption. In the present study, CF extract and its extract preparation process was optimized, micro-emulsion was developed,and pharmacodynamic evaluation was evaluated, and the following main results were obtained.(1)Optimization of CF extract preparation process: On the basis of single factor test, taking eugenol content of CF extract as index, Box-Behnken central composite design method was used to optimize the extracting process. The results showed that the best response for CF extract preparation was: 80% of ethanol concentration, extraction temperature 60 ℃, solid-liquid ratio 1:11, and extraction time 1.7 h. Under this condition, confirmatory test demonstrated the average phenol content of CF extract is 79.97%. This means that by optimizing response surface, CF extract preparation was stable and reliable.(2)Prescription optimization and preparation of CF extract microemulsion: By analyzing the solubility of CF extract in auxiliary materials, the pseudo-ternary phase diagrams were draw, microemulsion prescription was screened, the influences of the auxiliary materials adding sequence to microemulsion formation were investigated, and the CF extract microemulsion formulation and prepared method was determined. The results showed that ethyl oleate(EO)/ Crempphor RH 40(RH40) /1,2-propanediol/distilled water microemulsion system is easy to form, and stability is good. The optimized the microemulsion formulations were 1%, 3% or 5% CF extract(prescription 1, 2, 3, respectively), 27% RH40, 9% 1, 2-propanediol, 4% EO, 55%, 57% or 59% distilled water. Prepared method was that surfactant, co-surfactant and oil mixture took according to prescription, adding the prescribed amounts of CF extract stirred to dissolve and added the dropwise prescribed amount of distilled water was stirred. The prepared CF extract microemulsion was O/W type, yellow or brown clear and transparent, and good fluidity. The emulsion droplets were spherical distribution, and the average particle diameter of each prescription was 17.19, 18.53, 17.60 nm, respectively. The polydispersity indexes were 0.045, 0.154, 0.171, respectively, and the eugenol contents were 26.53, 34.26, 40.97mg/m L, respectively.(3)Pharmacodynamic study on CF extract microemulsion: To determine the in vitro aricidal activity of the CF extract microemulsion on rabbits itch mites, 0.5%-4.0%(r=0.5) CF extract microemulsion were used to immerse the rabbits itch mites. Under the microscope, the dead numbers of the experimental mites were recorded, the death rates were calculated to carry on Probit regression analysis, and the median lethal concentration(LC50) was calculated. The results showed that with concentration of 0.5%, 1%, 2%, 4% CF extract microemulsion at 24 h average mortality rate of the mites caused by were 11.7%, 48.3%, 60.0%, 88.3%, respectively, and by treatment of CF extract microemulsion for 24 h, LC50 for mites was 1.33%, and 95% confidence limits was 0.51%-3.02%. This means that CF extract microemulsion have high aricidal activity. |
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