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

Summary

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.

Abstract

As a traditional Chinese medicine (TCM), Epimedii folium (EF) has a history in medicine and food that is > 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 °C ± 10 °C, adding the crude EF, stir-frying it gently to 189 °C ± 10 °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.

Introduction

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 G....

Protocol

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 (Sieb. et Zucc.) Maxim. by researchers of The Institute of Biological Medicine, Chongqing Institute of Traditional Chinese Medicine.

1. Pr....

Results

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%, .......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetonitrile	Fisher	197164
Baohuoside (B )	Chengdu Manst Biotechnology Co., Ltd.	MUST-20042402
Chromatographic column	Waters Corporation	Symmetry C18
Design Expert software	Stat- Ease Inc., Minneapolis, MN	Trial Version8.0.6.1
Detector	Waters Corporation	2998
Disintegrator	Hefei Rongshida Small Household Appliance Co., Ltd.	S-FS553
Electronic analytical balance	Mettler-Toledo International Inc.	MS205DU
Epimedin A (EA)	Chengdu Manst Biotechnology Co., Ltd.	MUST-21112118
Epimedin B (EB)	Chengdu Manst Biotechnology Co., Ltd.	MUST-20080403
Epimedin C (EC)	Chengdu Manst Biotechnology Co., Ltd.	MUST-20080310
Ethanol	Chongqing Chuandong Chemical ( Group ) Co., Ltd.	20180801
Graphpad software	GraphPad Software Inc., San Diego, CA, USA	6.02
High Performance Liquid Chromatography (HPLC)	Waters Corporation	2695
Icariin	Chengdu Glip Biotechnology Co., Ltd.	21091401
Methanol	Chongqing Chuandong Chemical (Group) Co., Ltd.	20171101
Microporous membrane	Tianjin Jinteng Experimental Equipment Co., Ltd.	0.22μm
Mutton oil	Kuoshan Zhiniu Fresh Food Store	20211106
Office Excel office software	Microsoft	Office Excel 2021
Pharmacopoeia sieve	Shaoxing Shangyu Huafeng Hardware Instrument Co., Ltd.	R40/3
Pure water machine	Chongqing Andersen Environmental Protection Equipment Co., Ltd.	AT Sro 10A
Qualitative filter paper	Shanghai Leigu Instrument Co., Ltd.	18cm
Stereomicroscope	Carl Zeiss, Oberkochen, Germany	Stemi 2000
Ultrasonic cleaner	Branson Ultrasonics (Shanghai) Co.,Ltd.	BUG25-12
Zebrafish	China Zebrafish Resource Center (CZRC)	The AB strain