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 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 studies^5,6,7 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 extraction^5,6,7 and processing^9,10,11. 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.

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. Prepare the control product solution by accurately weighing the appropriate amount of each reference substance, namely, icariin, epimedin A (EA), epimedin B (EB), epimedin C (EC), and baohuoside I (BI), using an electronic analytical balance, and dissolve in methanol. Using these, prepare a mixed reference stock solution containing 381.61 µg/mL icariin, 124.14 µg/mL EA, 110.24 µg/mL EB, 1091.75 µg/mL EC, and 184.98 µg/mL BI.
2. Prepare the test product solution by crushing EF through a No. 3 sieve. Place approximately 0.2 g (using an electronic analytical balance) of crushed EF into a stoppered Erlenmeyer flask, add 20 mL of dilute ethanol, and then ultrasonicate at 400 W power and 50 kHz frequency for 1 h. Shake well, and pass through a 0.22 µm membrane filter to obtain the test solution.
3. Perform the chromatography as follows. Use high-performance liquid chromatography (HPLC) with a C₁₈ column with dimensions of 4.6 mm x 250 mm and an inner diameter of 5 µm. Use acetonitrile as mobile phase A and ultrapure water as mobile phase B. Use the following gradient elution parameters: 0-30 min, 24% A to 26% A; 30-31 min, 26% A to 45% A; 31-45 min, 45% A to 47% A. Use a detection wavelength of 220 nm (for the detector used, see Table of Materials). Keep the column temperature at 30 °C and the current velocity at 1.0 mL/min, and use a sample size of 10 µL.
4. To investigate the linear relationship, use the mixed reference solution as in step 1.1 diluted 2 times, 4 times, 8 times, 16 times, and 32 times, for icariin, EA, EB, EC, and BI, respectively. Use acetonitrile as mobile phase A and ultrapure water as mobile phase B.
5. Use the following gradient elution parameters: 0-30 min, 24% A to 26% A; 30-31 min, 26% A to 45% A; 31-45 min, 45% A to 47% A. Use a detection wavelength of 220 nm (for the detector used, see Table of Materials). Keep the column temperature at 30 °C and the current velocity at 1.0 mL/min and use a sample size of 10 µL. Finally, record the peak areas. Plot the linear regression with the reference concentration (x-axis, µg/mL) as the abscissa and the peak area (y-axis) as the ordinate using professional software (see Table of Materials).
6. Perform the precision test by measuring the mixed control solution six consecutive times by HPLC using the chromatographic conditions shown in step 1.3. Record the detection time and peak areas of each chemical composition, and calculate the relative standard deviations (RSD) of the peak areas to assess the precision (reproducibility) using the formula below:
   RSD% = standard deviation (SD)/arithmetic mean of calculated results (X) x 100 %
7. To perform the reproducibility test, accurately weigh the EF powder, and prepare six parts of the test product solution in parallel according to the method in step 1.2. Subject the prepared solutions to HPLC under the chromatographic conditions presented in step 1.3. Record the retention times and peak areas of each chemical composition and calculate the amounts of each compound from a standard curve (peak areas versus concentrations). Calculate the RSD% as above.
8. To perform the stability test, store the test solutions at room temperature, and measure their contents by the HPLC method described in step 1.3 at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h after preparation to assess the stability. Record the retention times and peak areas of each chemical composition and calculate the RSD% of the peak areas as above.
9. To perform the sample recovery test, weigh 0.2 g of EF powder into a stoppered Erlenmeyer flask for six replicates. Add an appropriate amount of the reference solution (the amount of reference substance added to the sample is equivalent to 100% of the known content of the sample) and prepare the test solution according to the method presented in step 1.2.
10. Inject the samples into the chromatograph and analyze according to the chromatographic conditions in step 1.3. Record the peak areas, and calculate the average recovery and RSD% values as below:
    Spiked sample recovery rate = (spiked sample content − sample content)/sample amount x 100%

2. Optimization of the EF mutton-oil processing technology using the Box-Behnken design-response surface methodology

1. Select the key parameters in EF processing, such as the amount of mutton oil (A; 15%-35%), the mutton oil temperature (B; 50-120 °C), and the frying temperature (C; 80-300 °C), as influential factors. Use the comprehensive scores of icariin, EA, EB, EC, and BI content as the evaluation indexes. The percentage of mutton oil here is the mass percentage.
2. Use the response surface analysis software (see Table of Materials) 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 3; set factors A, B, and C. Click on Continue. Set the Responses option to 1 (which was the comprehensive score). Click on Continue to complete the design. A total of 17 experiments were planned (see Table 1).
   NOTE: For the independent and dependent variables, along with their low, middle, and high levels, see Table 2.
3. Process the EF according to the specific parameters in Table 1; for example, for order number 1, weigh refined mutton oil as 15% v/v, and then heat to 50 °C to melt it. Add the crude EF to the melted mutton, stir-fry over a gentle fire (190 °C) until it is evenly shiny, and then remove and cool. Performed 17 experimental operations. A total of 17 groups of EF-processed products were obtained in this work.
   NOTE: Mutton oil is solid at room temperature (25 °C) and melts into liquid when heated. Mutton oil in a liquid state can be used as the excipient.
4. Prepare the test solutions of the processed products according to the method described in step 1.2. Then, analyze them using HPLC according to the chromatographic conditions described in step 1.3. 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. Use the comprehensive score calculation formula below to calculate the comprehensive scores of the 17 experimental groups:
   Comprehensive score = Z/Z_max × 0.5 + BI/BI_max × 0.5
   where Z is the sum of the icariin, EA, EB, and EC contents; Z_max is the maximum value of the sum of the icariin, EA, EB, and EC contents in the 17 experimental groups; BI is the BI content; and BI_max is the maximum value of the BI content in the 17 experimental groups.
5. Import the comprehensive scoring results for the 17 groups of experiments into the data analysis software (see Table of Materials) to analyze the experimental data. Under the evaluation items, select the quadratic process order option and polynomial model type option.

3. Testing the effect of processing on zebrafish embryonic development

1. Sample preparation
   1. Crush the crude and processed EF through a No. 3 sieve (see Table of Materials). To 100 g of each EF sample, add 1,000 mL of ultrapure water. Soak the EF for 0.5 h, boil the water twice for 30 min each, and then filter with filter paper.
   2. Combine the filtrates and concentrate the sample by heating. Add ultrapure water to a final volume of 100 mL to obtain the processed EF (PEF, 1 g/mL) and the crude EF (CEF,1 g/mL) stock solutions. Measure the amount of raw drug in each stock solution.
   3. Place aliquots of 1 mL, 1.5 mL, 2.5 mL, 5 mL, and 7.5 mL stock solutions in 10 mL volumetric flasks, and then add ultrapure water to volume to prepare the test solutions with concentrations of 100 mg/mL, 150 mg/mL, 200 mg/mL, 250 mg/mL, 500 mg/mL, and 750 mg/mL for the zebrafish embryotoxicity study.
      NOTE: The concentrations of the test solutions were prepared by referring to the relevant literature^20,21 and by performing preliminary experiments to give the 10-fold concentration gradient used in normal toxicology. CEF was an unprocessed sample, and PEF was a sample prepared with the best processing technology described in section 2.
2. Zebrafish husbandry and embryo treatment²¹
   1. Adapt wild-type zebrafish (see Table of Materials) at a controlled temperature for 2 days, keep them in a flow-through aquarium at pH 7.0-7.4 and feed them twice daily.
      NOTE: The inhibition of melanin formation in zebrafish was achieved by adding 1-phenyl-2-thiourea in a concentration of 0.003% (mass/volume) to the culture medium, which kept their bodies transparent for morphological observation.
   2. Select adult fertile wild-type zebrafish in the evening and separate them by using baffles in mating boxes. Remove the baffles the following morning, and allow the fish to spawn for 30 min. Collected the fertilized eggs with a dropper every 15 min. In total, 520 healthy wild-type embryos were collected. Keep the zebrafish embryos in an incubator at 28.5 °C for 24 h.
   3. Randomly assign the healthy embryos at 24 h post fertilization (hpf) to 13 groups, and along with one control group, separately soak in 10 mL of each of the following solutions in a culture dish: PEF: 100 µg/mL, 150 µg/mL, 200 µg/mL, 250 µg/mL, 500 µg/mL, 750 µg/mL; CEF: 100 µg/mL, 150 µg/mL, 200 µg/mL, 250 µg/mL, 500 µg/mL, 750 µg/mL . Treat the blank control group with the medium as a solution. Each group contained 40 embryos in this study.
      NOTE: The medium composition is 0.15 M NaCl, 5 mM KCl, 0.25 mM Na₂HPO₄, 0.45 mM KH₂PO₄, 1.3 mM CaCl₂, 1.0 mM MgSO₄, and 4 mM NaHCO₃.
   4. Culture the zebrafish in a constant temperature incubator for up to 120 hpf. Count the number of dead larvae every day, observe the main organ morphology of the larvae in each experimental group under a stereomicroscope (scale bar = 500 µm, see Table of Materials), and calculate the half-death concentration (LC₅₀) of zebrafish at 72 hpf by using data analysis software (see Table of Materials).

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

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