Name: optimization of the epimedii folium mutton-oil processing technology and testing its effect on zebrafish embryonic development
Uploaded: 2023-03-17T14:50:00
Description: Watch this Scientific Journal Video about Optimization of the Epimedii Folium Mutton-Oil Processing Technology and Testing Its Effect on Zebrafish Embryonic Development at JoVE.com

This work is supported by the Basic Scientific Research Business Project of Chongqing Academy of Traditional Chinese Medicine (Project Number: jbky20200013), the Performance Incentive Guidance Project of Chongqing Scientific Research Institutions (Project Number: cstc2021jxjl 130025), and the Chongqing Municipal Health Commission Key Discipline Construction Project of Chinese Materia Medica Processing.

All animal-related experiments were conducted with approval from the Experiment Ethics Committee of the Chongqing Institute of TCM (laboratory animal ethics review certificate number: ZJS2022-03).
1. Determination of the bioactive components
NOTE: The species used in this research was Epimedium sagittatum, and the samples were collected in Fengdu County, Chongqing. The sample was identified as a dry above-ground part of E. sagittatum...

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Independent variables and the determination of their levels 
The EF processing technology is only described in the 2020 edition of the Chinese Pharmacopoeia and the local Chinese medicine processing specifications published by 26 provinces, municipalities, and autonomous regions across the country1. The description involves the following steps: taking mutton oil and heating it to melt, adding EF shreds, stir-frying with a slow fire until it is uniform and shiny, taking it out...

Epimedii folium (EF) is the dried leaves of Epimedium brevicornu Maxim., Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Maxim., or Epimedium koreanum Nakai. EF can be used to treat osteoporosis, menopausal syndrome, breast lumps, hypertension, coronary heart disease, and other diseases1. As a traditional Chinese medicine (TCM), EF has a history in medicine and food of more than 2,000 years. Due to its low price and remarkable effect of tonifying the kidneys, it is widely used in medicines and health foods. EF is processed by stir-frying it with mutton oil, a process first described in Lei Gong Processing Theory written by Lei Xiao in the Liu Song period2. The efficacies of crude EF and stir-fried EF are quite different. Crude EF mainly dispels rheumatism, whereas the stir-fried EF warms the kidneys to reinforce yang3. At present, EF is widely used as a raw material in drugs and health foods; there are 399 listed Chinese patent medicines, nine imported health foods, and 455 domestic health foods with EF as a raw material4. This medicinal material has great application prospects. However, in recent years, there have been increasing reports of adverse reactions and human liver injury caused by health foods and Chinese patent medicines using EF as a raw material, and related toxicity studies5,6,7 have reported that EF as a raw material has potential safety risks.
Chinese medicinal processing refers to pharmaceutical techniques that can effectively reduce or eliminate the toxicity and improve the safety of TCMs. The traditional processing method of EF is stir-frying with mutton oil, which reduces the toxicity of EF and enhances its effect of warming the kidneys and promoting yang8. This processing method is included in the Chinese Pharmacopoeia and various processing specifications1. The process of EF is only specified as follows: for every 100 kg of EF, 20 kg amniotic oil (refined) is added, and it is mild-fired until uniform and shiny1. There are no strict EF processing method parameters in the above standards, so local processing specifications have not been unified to provide consistency. Therefore, it would be useful to conduct a systematic study of the EF process. In this paper, the Box-Behnken experimental design-response surface method was used to optimize the processing technology of EF.
The Box-Behnken experimental design is a method typically used to optimize the factors in a process. The extraction parameters can be optimized by establishing the functional relationship between multiple regression equation-fitting factors and effect values. Recently, this method has been widely used to study TCM extraction5,6,7 and processing9,10,11. Various studies have reported TCM preparation methods involving salt processing, wine processing, and stir-frying following a Box-Behnken design, such as for salt-processed Psoraleae fructus12, wine-processed Cnidii fructus13, and roasted Cinnamomi ramulus14. This method has reduced test time, high test accuracy, and is suitable for multi-factor and multi-level tests. The method is simpler than the orthogonal design test method and more comprehensive than the uniform design method15. The obtained relationships can determine the predicted value of any test point within the test range, which is a great advantage. A zebrafish model can be used to test whether EF is less toxic after processing.
In TCM toxicity studies, the zebrafish model has the dual advantages of the high throughput of cell experiments and the similarities with rodent experiments16. This model is characterized by its small size, high spawning rate, short reproduction cycle, and ease of breeding. The model can be used in large-scale synchronous experiments in cell culture plates, and the experimental drug dosage is small, the experimental cycle is short, the cost is low, and the whole experimental process is easy to observe and operate17. Zebrafish embryos are transparent and develop rapidly. Therefore, the toxicity and teratogenic effects of drugs on visceral tissues at different developmental stages can be directly observed under a microscope18. The gene homology between zebrafish and humans is as high as 85%18. The signal transduction pathway of zebrafish is similar to that of humans18. The biological structure and physiological function of zebrafish are highly similar to those of mammals18. Therefore, a zebrafish model for drug testing can provide experimental animals that are reliable and fully applicable to humans19.
In this study, we used the Box-Behnken design-response surface methodology to optimize the amount and temperature of mutton oil and the frying temperature used in the EF processing technology, with the contents of icariin, epimedin A, epimedin B, epimedin C, and baohuoside I as the evaluation indexes. The zebrafish model was used to preliminarily explore the effect of an EF water extract on zebrafish embryonic development before and after processing to evaluate the attenuation effect of processing on EF.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher</td>
			<td>197164</td>
			<td></td>
		</tr>
		<tr>
			<td>Baohuoside <img alt="Equation 1" src="/files/ftp_upload/65096/65096eq01.jpg" style="margin: auto;" /> (B<img alt="Equation 1" src="/files/ftp_upload/65096/65096eq01.jpg" style="margin: auto;" /> )&#160;</td>
			<td>Chengdu Manst Biotechnology Co., Ltd.</td>
			<td>MUST-20042402</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatographic column</td>
			<td>Waters Corporation</td>
			<td>Symmetry C18</td>
			<td></td>
		</tr>
		<tr>
			<td>Design Expert software</td>
			<td>Stat- Ease Inc., Minneapolis, MN</td>
			<td>Trial Version8.0.6.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Detector</td>
			<td>Waters Corporation</td>
			<td>2998</td>
			<td></td>
		</tr>
		<tr>
			<td>Disintegrator</td>
			<td>Hefei Rongshida Small Household Appliance Co., Ltd.</td>
			<td>S-FS553</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic analytical balance</td>
			<td>Mettler-Toledo International Inc.</td>
			<td>MS205DU</td>
			<td></td>
		</tr>
		<tr>
			<td>Epimedin A (EA)</td>
			<td>Chengdu Manst Biotechnology Co., Ltd.</td>
			<td>MUST-21112118</td>
			<td></td>
		</tr>
		<tr>
			<td>Epimedin B (EB)</td>
			<td>Chengdu Manst Biotechnology Co., Ltd.</td>
			<td>MUST-20080403</td>
			<td></td>
		</tr>
		<tr>
			<td>Epimedin C (EC)</td>
			<td>Chengdu Manst Biotechnology Co., Ltd.</td>
			<td>MUST-20080310</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Chongqing Chuandong Chemical ( Group ) Co., Ltd.</td>
			<td>20180801</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad software</td>
			<td>GraphPad Software Inc., San Diego, CA, USA</td>
			<td>6.02</td>
			<td></td>
		</tr>
		<tr>
			<td>High Performance Liquid Chromatography (HPLC)</td>
			<td>Waters Corporation</td>
			<td>2695</td>
			<td></td>
		</tr>
		<tr>
			<td>Icariin</td>
			<td>Chengdu Glip Biotechnology Co., Ltd.</td>
			<td>21091401</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Chongqing Chuandong Chemical (Group) Co., Ltd.</td>
			<td>20171101</td>
			<td></td>
		</tr>
		<tr>
			<td>Microporous membrane</td>
			<td>Tianjin Jinteng Experimental Equipment Co., Ltd.</td>
			<td>0.22&#956;m</td>
			<td></td>
		</tr>
		<tr>
			<td>Mutton oil</td>
			<td>Kuoshan Zhiniu Fresh Food Store</td>
			<td>20211106</td>
			<td></td>
		</tr>
		<tr>
			<td>Office Excel office software</td>
			<td>Microsoft</td>
			<td>Office Excel 2021</td>
			<td></td>
		</tr>
		<tr>
			<td>Pharmacopoeia sieve</td>
			<td>Shaoxing Shangyu Huafeng Hardware Instrument Co., Ltd.</td>
			<td>R40/3</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure water machine</td>
			<td>Chongqing Andersen Environmental Protection Equipment Co., Ltd.</td>
			<td>AT Sro 10A</td>
			<td></td>
		</tr>
		<tr>
			<td>Qualitative filter paper</td>
			<td>Shanghai Leigu Instrument Co., Ltd.</td>
			<td>18cm</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Carl Zeiss, Oberkochen, Germany</td>
			<td>Stemi 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>Branson Ultrasonics (Shanghai) Co.,Ltd.</td>
			<td>BUG25-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Zebrafish</td>
			<td>China Zebrafish Resource Center (CZRC)</td>
			<td>The AB strain</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimization of the epimedii folium mutton-oil processing technology and testing its effect on zebrafish embryonic development

As a traditional Chinese medicine (TCM), Epimedii folium (EF) has a history in medicine and food that is &#62; 2,000 years old. Clinically, EF processed with mutton oil is often used as a medicine. In recent years, reports of safety risks and adverse reactions of products that use EF as a raw material have gradually increased. Processing can effectively improve the safety of TCM. According to TCM theory, mutton-oil processing can reduce the toxicity of EF and enhance its tonifying effect on the kidneys. However, there is a lack of systematic research and evaluation of EF mutton-oil processing technology. In this study, we used the Box-Behnken experimental design-response surface methodology to optimize the key parameters of the processing technology by assessing the contents of multiple components. The results showed that the optimal mutton-oil processing technology of EF was as follows: heating the mutton oil at 120 &#176;C &#177; 10 &#176;C, adding the crude EF, stir-frying it gently to 189 &#176;C &#177; 10 &#176;C until it is evenly shiny, and then removing it and cool. For every 100 kg of EF, 15 kg of mutton oil should be used. The toxicities and teratogenicities of an aqueous extract of crude and mutton-oil processed EF were compared in a zebrafish embryo developmental model. The results showed that the crude herb group was more likely to cause zebrafish deformities, and its half-maximal lethal EF concentration was lower. In conclusion, the optimized mutton-oil processing technology was stable and reliable, with good repeatability. At a certain dose, the aqueous extract of EF was toxic to the development of zebrafish embryos, and the toxicity was stronger for the crude drug than for the processed drug. The results showed that mutton-oil processing reduced the toxicity of crude EF. These findings can be used to improve the quality, uniformity, and clinical safety of mutton oil-processed EF.

Methodological investigation results 
A linear relationship between the concentration of icariin, EA, EB, EC, BI, and chromatographic peak areas was observed (see Table 3). The RSD% values (n = 6) of the chromatographic peak areas of icariin, EA, EB, EC, and BI were 0.28%, 1.22%, 0.65%, 1.67%, and 1.06%, respectively, indicating that the precision of the HPLC measurements was good. The RSD% values (n = 6) of the contents of icariin, EA, EB, EC, and BI were 1.59%, 1.46%, 1.86%, 2.29%...

Watch this Scientific Journal Video about Optimization of the Epimedii Folium Mutton-Oil Processing Technology and Testing Its Effect on Zebrafish Embryonic Development at JoVE.com

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.

As a traditional Chinese medicine (TCM), Epimedii folium (EF) has a history in medicine and food that is &#62; 2,000 years old. Clinically, EF processed ...

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.

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