Optimization of the Epimedii Folium Mutton-Oil Processing Technology and Testing Its Effect on Zebrafish Embryonic Development

In this protocol, the mutton-oil processing technology of Epimedii folium (EF) was optimized by applying a Box-Behnken experimental design-response surface methodology, and the effect of crude and optimized water-extracted EF on zebrafish embryonic development was preliminarily investigated.

In this study, we adopted a widely used processing method and acquired a stable and reliable persistent technique. The result showed that the toxicity of processed Epimedii was reduced. Response surface method requires fewer experiments and shorter cycles, which cannot only avoid the problems existing in traditional mathematical statistic, but also help users to select the optimal conditions.

This method can also be widely used in the chemical industry, biologic engineering, food industry, Chinese medicine preparation, Chinese medicine extraction, and so on. After preparing the solution and performing the chromatography, select the key parameters in EF processing, such as the amount of mutton oil, the mutton oil temperature, and the frying temperature as influential factors. Use the comprehensive scores of icariin, EA, EB, EC, and BI content as the evaluation indexes.

Use the response surface analysis software to design the Box-Behnken response surface experiments, explore the quadratic response surface, and construct a second order polynomial model. Select the new Box-Behnken design, and set the numeric factors option to three. Set factors A, B, and C.Click on continue, then set the responses option to one, and click on continue to complete the design.

Process the EF according to the specific parameters, for example, for order number one, where refined mutton oil as 15%volume by volume, and then heat it to 50 degrees Celsius to melt it. Add the crude EF to the melted mutton. Stir fry over a gentle fire until it is evenly shiny, then remove and cool the EF.Prepare the test solutions of the processed products, and analyze them using HPLC as described in the manuscript.

Record the retention times and peak areas of each chemical composition, and calculate the contents of the icariin, EA, EB, EC, and BI in each test solution against an external standard curve. Calculate the comprehensive scores of the 17 experimental groups using the comprehensive score calculation formula. Import the comprehensive scoring results for the 17 groups of experiments into the data analysis software to analyze the experimental data.

Under the evaluation items, select the quadratic process order option and polynomial model type option. The response surface of the mutton oil amount and the frying temperature was steeper compared to the other tested factors, and the contour lines tended to be more elliptical, indicating that the interaction between these two factors was more significant than the interactions between other factors. The HPLC chromatogram of the mixed reference substance, crude Epimedii Folium and Epimedii Folium processed products demonstrate that the BI content in raw EF is low, while it increases after processing.

The crude group shows obvious spinal deformities, body curvature, pericardial edema, and liver deformation at 150 micrograms per milliliter drug concentration, whereas those changes were rare in juvenile fish in the processed group. All zebrafish in the crude drug group died 24 hours after 200 micrograms per milliliter drug administration, whereas the mortality in the processed group was only 6.67%Administration of 200 micrograms per milliliter of EF caused the death of all zebrafish in the crude drug group after 72 hours, while in the processed group, 500 micrograms per milliliter of EF caused the death of all zebrafish. The median lethal concentration of the two experimental groups at 72 hours post fertilization showed that the LC 50 was 151.3 micrograms per milliliter in the crude group, and 219.8 micrograms per milliliter in the processed group.

The most important thing in the experiment is selecting factors of the response surface method, and determine the parameters of these factors.