| Ginseng is a traditional Chinese medicine with the effect of medicine.Ginsenoside is one of the main effective substance of ginseng,and the various components have the effect of improving the learning and memory disorder,sedation and excitement of rats.These components contain ginsenoside Re,ginsenoside Rg2 and so on.A variety of ingredients of Ginsenosides include sedative effect,and the classification of ginsenoside Rg has the effect of excitement.Hu also found that ginsenoside and W/O vaccine have a suitable synergistic effect,then significantly improve the body’s immune response.Preparations containing Ginsenoside were mostly oral solution,which are easy to operate during inoculation while have some disadvantages as inferior immune effect.The purpose of this study is to prepare a W/O/W ginsenoside double emulsion in order to reduce the side effects and toxicity of drugs in the vaccine,so as to reduce the times and dosage of injection to improve the immune effect.The double emulsions include W/O/W and O/W/O,and the W/O/W ginsenoside double emulsion is a new type of preparation.In this paper,the preparation of ginsenoside double emulsion,the research of high-performance liquid quantitative,the qualitative research of thin-layer chromatography,and the research of stability are evaluated in order to develop a new injection vaccine with convenient operation,controllable quality and effective immune.Experiment I Preparation and structure stability of W/O/W Ginsenoside double emulsion.The preparation process of W/O/W ginsenoside double emulsion was optimized by repetitive experiments.The effects of various factors on the viscosity,appearance,preparation,particle size and stability of W/O/W ginsenoside double emulsion were investigated.The results showed that the concentration of ginsenoside in the aqueous phase,the emulsifying strength and the emulsifying time are the main factors to influence the preparation process and quality.So these three factors are chosen for orthogonal test and optimization.The results showed that ginsenoside solution and adjuvant reach the optimal conditions under the mass ratio of 1:1,or volume ratio of 4.6:5.4,emulsification of 30 minutes and 300 rpm with normal viscosity and excellent stability and other characteristics.Then determined the morphology of W/O/W ginsenoside double emulsion,and found creamy yellow in appearance and light blue when shaking.The preparation is liquid with cloudy diffusion.Finally,the structure of W/O/W ginsenoside double emulsion is investigate by transmission electron microscopy(TEM),which present that the particle size was in the range of 100-200 nm.It was found that the structural stability of the W/O/W ginsenoside double emulsion was higher than that without ginsenosides,indicating that the integration and structural stability during ginsenosides and adjuvant are favorable.The stability structure of the W/O/W ginsenoside double emulsion provides a basis for a wide range of clinical applications for other ginsenoside preparation,which opens up a broader space for the application of double emulsion.Experiment II Qualitative examination of W/O/W Ginsenoside double emulsion.First,according to the identification of ginsenoside Re,Rgl and Rd in the veterinary pharmacopoeia,the methanol and ethyl acetate-chloroform-water(11:20:7.5:5)was used as the developing solvent to remove,dry,and sprayed with chromogenic agent in the 105 ℃drying closet heated to emerge the spot color.To identify three monomer components(Re,Rgl and Rd)in the W/O/W ginsenoside double emulsion,the results show that in the chromatogram of the test sample,the corresponding position of ginsenoside reference showed the same color of spots,while absent in the negative reference.Then the four kinds of sampling volume of the chromatographic effect are compared.The results showed that the color of the spot gradually increased with the increase of the amount of the sample,and the spot reach the clearest when the sampling volume was 4 μl.However,considering the expensive price of ginseng,the optimum sampling volume is 2 μl.Finally,with the establishment of the ideal detection method in accordance with the requirements in veterinary pharmacopoeia,the three batches of pilot products of W/O/W ginsenoside double emulsion were identified and inspected.Experiment III Quantitative examination of W/O/W Ginsenoside double emulsion.First,the detection conditions of three kinds of monomer components in W/O/W ginsenoside double emulsion were determined by HPLC.Then,18 kinds of isometric and 3 kinds of gradient elution detection methods are established under the optimal conditions,and the precision,recovery rate,systematic adaptability and linear relationship are evaluated.The results were as follows:gradient elution conditions are 0.1%phosphoric acid solution:acetonitrile(0-30 min,81:19;30-45 min,76:24;45-85 min,60:40);column temperature is 30.The prepared W/O/W ginsenoside double emulsion is optimized with high precision and recovery rate,and favourable system adaptability and linear relationship.The contents of Rgl,Re and Rd in W/O/W ginsenoside double emulsion are 18.26 μg to 25.72 μg,28.67 μg to 35.46 μg and 17.28 μg to 24.18 μg,respectively.Consistent with the pharmacopoeia of content requirements on ginsenoside Rg1,Re and Rd,indicating well established detection method.Experiment IV Stability study of W/O/W Ginsenoside double emulsion.Light acceleration(Light 0 d,light 5 d,light 10 d,illumination 4500 lx),4 ℃,temperature and humidity accelerated(30±2 ℃,relative humidity 65%±5%)and room tempreture are determined using a well established HPLC method to explore the contents of three monomer components of Rgl,Re and Rd in W/O/W ginsenoside double emulsion.In the light(5 d)stability test,the content of ginsenoside Rgl was between 17.70 and 22.72 mg/ml,the content of Re was between 27.96 and 35.46 mg/ml,and the content of Rd was between 17.17 and 22.16 mg/ml.In the light(10 d)stability test,the content of ginsenoside Rgl was between 17.25 and 20.35 mg/ml,the content of Re was between 26.19 and 33.02 mg/ml,and the content of Rd was between 16.07 and 20.14 mg/ml.In the stability test(4 ℃,place for one month),the content of ginsenoside Rgl was between 18.01 and 24.87 mg/ml,the content of Re was between 28.17 and 36.00 mg/ml,and the content of Rd was between 17.32 and 24.64 mg/ml.In the temperature and humidity(place for one month)accelerated stability test,the content of ginsenoside Rgl was between 15.33～17.86 mg/ml,content of Re was between 24.07～30.10 mg/ml and content of Rd was between 14.06～18.65 mg/ml.At room temperature stability(place for one month)test:content of ginsenoside Rg1 was between 11.36～14.72 mg/ml,content of Re was between 19.43～23.14 mg/ml,content of Rd was between 10.30～14.47 mg/ml.The results showed that the above parameters of the three monomer components of W/O/W ginsenoside double emulsion had no effect at 4℃ with well index value.The effect of light acceleration was higher than 4 ℃,followed by temperature and humidity acceleration,and the three components of W/O/W ginsenoside double emulsion have the greatest effect at room temperature.The above test results show that the best storage conditions of W/O/W ginsenoside double emulsion are 4 ℃,dry and dark,and avoiding high temperature and humidity. |
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