Preparation And Quality Evaluation Of Toad Skin Extract Loaded Solid Lipid Nanoparticles

Objective: There are a variety of effective chemical constituents in dry skin of Bufo bufo gargarizans Cantor collectively known as bufogenin, a class of polycyclic compounds within the same steroid skeleton of the ingredients, including cinobufagin, resibufogenin and bufalin. It is widely used in clinic for its pharmacologic actions of cardiotonic, antitumor,local anesthesia,analgesia and anti-inflammatory. But Currently forms used in the clinic are traditional tablets, oral liquid, capsules and injections,But the amount of medicine is large,patients have less compliance; Oral stench has poor taste; stimulate intravenous injections. With the development of science, new formulations due to its widely advantages have been widely researched, therefore, it is necessary to research new dosage form for toad skin extract to improve the existing inadequate dosage. Solid lipid nanoparticles(SLN) are a new pharmaceutical delivery system,which are typically spherical with an average diameter between 50 to 1000 nanometers.SLN generally prepared by using solid natural or synthetic lipids such as glyceryl monostearate, lecithin, triglycerides as the carrier, the drug encapsulated in lipid core through different method. It has been proposed that SLNs combine numerous advantages over the other colloidal carriers i.e. incorporation of no biotoxicity of the carrier, increased drug stability, possibility of controlled drug release and drug targeting and no problems with respect to large scale production. This paper intends to optimize the extraction of effective chemical constituents from toad skin, and prepare toad skin extract loaded Solid lipid nanoparticles, the purpose is to control the drug release and drug targeting, increase the stability of the drug, high drug loading rate, enhance the patients on medication compliance.Methods: HPLC method were established to determinate the content ofcinobufagin and resibufogenin in toad skin extract, the test method were validated. Optimization of extraction process of toad skin, investigation of the influence of drying toad skin soak time effect on the extraction rate, water extraction and alcohol precipitation and ethanol boiling reflux method had the merits of toad skin extract. Alcohol precipitation and ethanol boiling reflux method was used for toad skin extraction and orthogonal test of three factors-three levels was used for the optimization procedure taking ethanol concentration,decoction time and decoction times as independent variables,taking yield of extract and content of cinobufagin and resibufogenin as the response variables. Synthetic weighted mark method was taken to value the result, the optimization technology was validated. To evaluate the quality of the toad skin extract, thin layer chromatography was used for the qualitative identification of extracts, HPLC method was established to determinate the content of cinobufagin and resibufogenin in toad skin extracts. Based on the single factor experiment, the single factor investigation the influence of the adding sequence of oil-water phase, adding method of oil phase, ultrasound conditions, emulsifying time, cooling method, external water phase dispersion type and concentration ratio, agent dosage and the ethanol concentration in oil phase on formulation and preparation process, preparation process of toad skin extract loaded solid lipid nanoparticles was optimized by uniform design. To evaluate the quality of the toad skin extract loaded solid lipid nanoparticles, microscope was used to observe nanoparticles morphology and particle size,and the entrapment efficiency and drug loading of cinobufagin and resibufogenin were measured. The stability study was operated by placing the nanoparticles in 60 ℃, 25 ℃, 4 ℃, sampling and determination of encapsulation efficiency in 0d,3d,6d,12 d,24d, respectively.Results: Standard curve equation of cinobufagin: A=36793C+743.9( r=0.9990, n=6). Standard curve equation of resibufogenin: A=28149C+2084.5(r=0.9990,n=6). The intra-day precision RSD of low,medium and high concentrations for cinobufagin and resibufogenin were 2.95%、1.04%、2.92% and 2.92%、3.03%、2.41%, respectively; The inter-day precision RSD were 6.28%、1.96%、2.67% and 6.18%、3.51%、2.78%, respectively.The average recovery rate cinobufagin and resibufogenin were 97.77% and 98.31%.RSD of 24 h peak area for cinobufagin and resibufogenin were 1.079% and 2.836%. Screening of alcohol decocting reflux method of orthogonal test, the optimum extraction process was obtained: 10 times volume of 80% ethanol, three times of decoction, 20 min per time. The TLC results showed that contain cinobufagin and resibufogenin income extract. The average value of toad skin extract of cinobufagin and resibufogenin content were cinobufagin 2.124 mg·g-1, resibufogenin 1.122 mg·g-1. The single factor investigation according to the entrapment efficiency and drug loading: the oil phase into the aqueous phase was significantly better in the aqueous phase is added into the oil phase, and drops into the height selection for the liquid surface is about 5 cm, slowly dropping. Ultrasonic conditions as: power 400 W, intermittent 20 s, total 4min, emulsifying time is 150 min. Cooling method of cooling the choice of the first ice bath stirring 15 min and then transfer the refrigerator. The selection of P188 and Tween-80 mixed dispersant(5:1 ratio), nanoparticles have good stability, uniform distribution of nanoparticles was observed under the microscope. The optimized by uniform design test preparation of toad skin extract nanoparticles,the optimal prescription for the ATO 100 mg, soybean lecithin 300 mg, drug 300 mg is added to the 5%P188 42 ml and 0.5%TWeen-80 8ml emulsification and 150 min agent. According to the optimization of solid lipid nanoparticle formulation of milky white, morphology was observed by transmission electron microscope, visible the particle size uniform, spherical or ellipsoidal, laser scattering particle size analyzer measured the average particle size of(138.5 + 4.2) nm, 90% particle size less than(208.1 + 27.9) nm, the polydispersity index was0.143±0.023. Results of the stability studies show, with the increase of temperature and the placement of the extension of time, the entrapment efficiency of toad skin extract nanoparticles decreased gradually, the color from milk white to pale yellow, of which 4℃ placed 24 d encapsulation rate is above 80%.Conclusions: HPLC to determine the content of cinobufagin and resibufogenin was specificity and reliable. The concentration of ethanol is the main factor in toad skin fat soluble component extraction process, orthogonal design test shows that the method extract rate is very high, the content of effective components is high, good reproducibility, a multi index comprehensive evaluation method for the optimization of toad liposoluble components extraction process is reasonable, feasible. The experiment of preparation of toad skin extract loaded solid lipid nanoparticles with very good encapsulation efficiency and drug loading, and small particle size distribution, preparation technology has a good reproducibility and stability, to establish a stable and reliable quality control standards. Toad skin extract loaded solid lipid nanoparticles should be under the condition of low temperature preservation.