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 (Sieb. et Zucc.) Maxim. by researchers of The Institute of Biological Medicine, Chongqing Institute of Traditional Chinese Medicine.
<ol>
	<li>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 &#181;g/mL icariin, 124.14 &#181;g/mL EA, 110.24 &#181;g/mL EB, 1091.75 &#181;g/mL EC, and 184.98 &#181;g/mL BI.</li>
	<li>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 &#181;m membrane filter to obtain the test solution.</li>
	<li>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 &#181;m. 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 &#176;C and the current velocity at 1.0 mL/min, and use a sample size of 10 &#181;L.</li>
	<li>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.</li>
	<li>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 &#176;C and the current velocity at 1.0 mL/min and use a sample size of 10 &#181;L. Finally, record the peak areas. Plot the linear regression with the reference concentration (x-axis, &#181;g/mL) as the abscissa and the peak area (y-axis) as the ordinate using professional software (see Table of Materials).</li>
	<li>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 %</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#8722; sample content)/sample amount x 100%</li>
</ol>
2. Optimization of the EF mutton-oil processing technology using the Box-Behnken design-response surface methodology
<ol>
	<li>Select the key parameters in EF processing, such as the amount of mutton oil (A; 15%-35%), the mutton oil temperature (B; 50-120 &#176;C), and the frying temperature (C; 80-300 &#176;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.</li>
	<li>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.</li>
	<li>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 &#176;C to melt it. Add the crude EF to the melted mutton, stir-fry over a gentle fire (190 &#176;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 &#176;C) and melts into liquid when heated. Mutton oil in a liquid state can be used as the excipient.</li>
	<li>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/Zmax &#215; 0.5 + BI/BImax &#215; 0.5 
	where Z is the sum of the icariin, EA, EB, and EC contents; Zmax 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 BImax is the maximum value of the BI content in the 17 experimental groups.</li>
	<li>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.</li>
</ol>
3. Testing the effect of processing on zebrafish embryonic development
<ol>
	<li>Sample preparation
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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 literature20,21 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.</li>
	</ol>
	</li>
	<li>Zebrafish husbandry and embryo treatment21
	<ol>
		<li>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.</li>
		<li>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 &#176;C for 24 h.</li>
		<li>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 &#181;g/mL, 150 &#181;g/mL, 200 &#181;g/mL, 250 &#181;g/mL, 500 &#181;g/mL, 750 &#181;g/mL; CEF: 100 &#181;g/mL, 150 &#181;g/mL, 200 &#181;g/mL, 250 &#181;g/mL, 500 &#181;g/mL, 750 &#181;g/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.</li>
		<li>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 &#181;m, 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).</li>
	</ol>
	</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+modern+pharmacological+effects+of+traditional+Chinese+medicine+for+tonifying+kidney+yang.">Study on modern pharmacological effects of traditional Chinese medicine for tonifying kidney yang.</a> Journal of Hubei University of Chinese Medicine. 13 (04), 63-66 (2011).</li><li>Luo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+chemical+constituents+and+pharmacological+studies+of+Epimedium.">Advances in the chemical constituents and pharmacological studies of Epimedium.</a> Asia-Pacific Traditional Medicine. 15 (6), 190-194 (2019).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+icariin+on+ER&#946;+gene+expression+and+serum+estradiol+level+in+ovariectomized+rats.">Effects of icariin on ER&#946; gene expression and serum estradiol level in ovariectomized rats.</a> Hunan Journal of Traditional Chinese Medicine. 32 (1), 150-152 (2016).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+epimedin+A+on+osteoclasts+and+osteoporotic+male+mice.">Effects of epimedin A on osteoclasts and osteoporotic male mice.</a> Chinese Journal of Veterinary Science. 41 (07), 1359-1364 (2021).</li><li>Liu, Y. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+icariin+and+epimedium+C+on+microstructure+of+bone+tissue+in+glucocorticoid+osteoporosis+model+mice+based+on+Micro-CT+technique.">Effects of icariin and epimedium C on microstructure of bone tissue in glucocorticoid osteoporosis model mice based on Micro-CT technique.</a> Drug Evaluation Research. 43 (09), 1733-1739 (2020).</li><li>Zhan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+antiosteoporotic+activity+for+micro+amount+icariin+and+epimedin+B+based+on+the+osteoporosis+model+using+zebrafish.">Evaluation of antiosteoporotic activity for micro amount icariin and epimedin B based on the osteoporosis model using zebrafish.</a> Chinese Pharmaceutical Journal. (24), 30-35 (2014).</li><li>Zhan, Y., Wei, Y. -. J., Sun, E., Xu, F. -. J., Jia, X. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+zebrafish+model+combined+with+hyphenated+chromatographic+techniques+for+evaluation+anti-osteoporosis+activity+of+epimendin+A+and+its+metabolite+baohuoside+I.">Two-dimensional zebrafish model combined with hyphenated chromatographic techniques for evaluation anti-osteoporosis activity of epimendin A and its metabolite baohuoside I.</a> Acta Pharmaceutica Sinica. 49 (06), 932-937 (2014).</li></ol>

The authors declare no conflicts of interest.

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, and letting it cool. Additionally, 20 kg (abbreviated as 20%) of mutton oil (refined) is used for every 100 kg of Epimedium. However, the parameters of the processing process of EF are not specified. Among the independent variables in this experiment, three key factors could be quantified in the production process: the mutton oil dosage, the mutton oil temperature, and the frying temperature. The value range should be set according to the above description. From the results of the preliminary test, it can be seen that when the amount of mutton oil is 15%, the EF leaves can be evenly coated with mutton oil. When the dosage exceeds 35%, there is too much mutton oil. Finally, the range of the mutton oil amount should be 15%-35%. When the temperature reaches 50 &#176;C, mutton oil melts. When the temperature reaches &#62;120 &#176;C, the mutton oil starts to smoke, and the temperature is too high. Therefore, the temperature range of the mutton oil should be 50 &#176;C-120 &#176;C. The Chinese Pharmacopoeia 2020 edition stipulates that EF should be stir-fried with a slow fire. The slow fire should not exceed 200 &#176;C, and the frying temperature should range from 80 &#176;C-300 &#176;C.
Comprehensive scoring 
During the processing of Epimedium, the glycosidic bonds are broken, and the glycosidic components are transformed into lower glycosidic components. The determination of the content of EF-processed products in the 2020 edition of the Chinese Pharmacopoeia is based on the determination of the total content of icariin, EA, EB, and EC in the original medicinal materials, and a monoglycoside component, BI, is listed separately as an indicator. In this experiment, the total weight of the icariin, EA, EB, and EC in the processed EF was 50%, the weight of the BI was 50%, and the comprehensive score was set based on these values.
Response surface methodology (RSM) is a statistical technique for identifying optimal process parameters and solving multivariate problems. In this technique, a reasonable experimental design is used to obtain certain data through experiments, and a multivariate quadratic regression equation is used to derive a functional relationship between the factors and responses22. Uniform design and orthogonal design process optimization are commonly used, but their test accuracy is not high, and the mathematical model is not very predictable. The mathematical model underlying RSM is highly predictable. RSM requires fewer experiments and shorter cycles, which not only can eliminate the problems associated with traditional mathematical statistics but can also clarify the relationships between factors and responses23. RSM conceptualizes the system&#39;s response as a function of one or more factors and uses graphical techniques to display this functional relationship to help the user to select the optimal conditions in the experimental design by intuitive visual observation. These advantages have led to the wide use of this method in the chemical industry24, biological engineering, the food industry25, the pharmaceutical industry, and TCM preparations.
Although RSM can identify the functional relationship between responses (indexes to be investigated) and factors (independent variables), not all experiments are suitable for response surface optimization because there is not always a strong functional relationship between the response and factors. RSM often obtains continuous function relationships, which require all the factors to be continuous variables. Nevertheless, not all factors to be investigated are continuous variables or have significant effects on the response values at the beginning of the experimental design. To reduce the number of experiments and improve the accuracy of the response surface modeling, screening is necessary to select the significant factors and determine their levels through factorial design, uniform design, or orthogonal design before conducting response surface design methodology. The biggest advantage of response surface methodology is that once the model is correctly established, the response value under any combination of conditions can be predicted, and the functional relationship can be seen more intuitively and visually through the 3D response surface. This visualization is of great help to researchers in finding the optimal processing conditions26.
This study used the Box-Behnken design principle of RSM to design 17 combined experiments using the comprehensive chemical content score of EF as the response value. Finally, the best process optimization results were obtained by regression analysis. The processing technology was optimized as follows: heating the mutton oil at 120 &#176;C &#177; 10 &#176;C, adding the crude EF, frying it with a gentle fire (189 &#176;C &#177; 10 &#176;C) until it is evenly shiny, and then removing and cooling it. For every 100 kg of EF, 15 kg of mutton oil (refined oil) should be used. Our results showed that the EF process was stable, reliable, and repeatable. Additionally, factor interactions were analyzed, and the interaction between the mutton oil amount and frying temperature, but not the interactions between other factors, was significant. This study demonstrated that the response surface design, as a method to analyze the interactions between factors and the relationships between factors and their response surface values, enabled the optimization of the processing conditions in a short period with a minimal number of experiments. The selected factors in this study were the key factors identified in the single-factor screening experiment, and their levels were determined in a preliminary experiment. The test samples conformed to the characteristics of the response surface method, so the study was able to use the response surface methodology to establish a predictive model. The experimental results can provide a reference for improving the quality and uniformity of the processed EF.
Zebrafish embryos are used as model organisms in the field of developmental genetics because they are transparent, develop in vitro, and are easy to observe27. Commonly used zebrafish toxicity indicators in developmental toxicity studies include embryonic mortality, embryonic malformation rate, yolk-sac edema, pigment formation, egg condensation, tail extension, head morphology, and body-segment formation, among others28. Compared with mammalian toxicity evaluation techniques, the specificity of zebrafish embryos for compound toxicity detection is 70%-80%, and the sensitivity exceeds 80%18. Ton et al.29 found that the accuracy of evaluating the developmental toxicity of non-teratogenic compounds with zebrafish embryos was 75%. Teratogenic compounds were evaluated with 100% accuracy here. Although TCM has the characteristics of complex components and unclear target organs of toxicity, zebrafish embryos can still be used as an experimental animal model for the accurate and rapid evaluation of developmental toxicity. He et al.30 found that emodin affected the survival and hatching rates of zebrafish embryos, causing trunk bending and yolk-sac edema. Chen et al.31 found that muscone caused zebrafish embryo pericardial edema, spinal curvature, and yolk-sac edema. He et al.32 found that Arnebiae Radix had lethal effects on zebrafish at all developmental stages, and 1.0 mg/L Arnebiae Radix inhibited embryonic development, resulting in a reduced number of somites, tail deformities, body bending, and reduced melanin in zebrafish embryos.
To investigate the effects of crude and processed EF on zebrafish embryo development, a zebrafish embryo developmental toxicity experiment was conducted in this study. The data showed that the LC50 values were 151.3 &#181;g/mL for the crude group (S) and 219.8 &#181;g/mL for the processed group (P). Observing the zebrafish bodies in each experimental group through a microscope showed an obvious degree of zebrafish teratogenicity in the crude group. Most of the fish showed varying degrees of teratogenicity, including spinal deformity, body curvature deformity, pericardial edema, swim bladder incompleteness, or liver deformation, and these observations were rare in the processed group. These experiments showed that the toxicity of EF was significantly reduced after processing, suggesting that processing could reduce drug toxicity in humans. The experimental results provide a reference for improving the clinical medication safety of mutton oil-processed EF.
Traditional Chinese medicine suggests that the function of the kidney is closely related to the growth, development, and reproduction of the human body33. The ancient books of traditional Chinese medicine record that the kidney is the bone marrow of the body. The kidney stores essence, and the marrow resides in the bone cavity to nourish the bone. When the kidney essence is deficient, the bone marrow is reduced34. The traditional Chinese medicine of tonifying the kidney yang can treat lumbar debility, osteoporosis, impotence, premature ejaculation, and uterine cold infertility35. EF is one of the representative medicinal materials for tonifying the kidney yang. Modern pharmacological studies have shown that EF has obvious effects on the skeletal system, immune system, reproductive system, cardiovascular system, and nervous system, as well as having anti-tumor effects36. In terms of activity on the skeletal system, icariin37 can improve the level of serum E2 in ovariectomized rats and upregulate the expression of ER&#946; mRNA in the bone tissue of ovariectomized rats. The synthesis of ER&#946; is increased, thereby improving the biological effect of ER, weakening the bone resorption activity of osteoclasts, and enhancing the bone formation of osteoblasts. The changes in bone resorption are greater than the negative balance of bone metabolism. Epimedin A can improve the bone microstructure and serum bone turnover markers in osteoporosis model mice by inhibiting osteoclast formation, differentiation, and bone resorption and playing a role in bone protection38. Epimedin C has obvious anti-osteoporosis activity, mainly in terms of increasing the bone mass and improving the trabecular microstructure to ultimately increase bone strength39. Other studies have shown that epimedin B40 and baohuoside I41 have anti-osteoporosis activity.

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%, and 0.98%, respectively, indicating that the method had good repeatability. The RSD% values (n = 6) of the peak areas of icariin, EA, EB, EC, and BI in the samples were 1.49%, 1.96%, 1.42%, 0.96%, and 0.81%, respectively, indicating that the sample solution was stable within 24 h. The average recovery rates of icariin, EA, EB, EC, and BI were 99.98%, 100.14%, 100.09%, 100.75%, and 100.94%, respectively, and the RSD% values were 0.56%, 0.78%, 0.84%, 1.10%, and 1.47%, respectively (see Table 4). These results show that the accuracy of the method met the requirements.
The above experimental results showed that the analytical method provided results that had excellent precision, reproducibility, and accuracy and were acceptable for the quality analysis of the EF-processed products.
Optimization of the mutton-oil processing technology of EF by applying the Box-Behnken design-response surface methodology 
We performed quadratic polynomial regression fitting of the above data to obtain the following model: Y = 0.86 &#8722; 0.11 x A + 0.025 x B &#8722; 0.078 x C &#8722; 0.023 x A x B &#8722; 0.037 x A x C + 0.037 x B x C &#8722; 0.045 x A2 + 2.5 x 10-3 x B2 &#8722; 0.14 x C2. The variance analysis gave a value of P &#60; 0.01, indicating that the model was significant. The P value of the lack of fit was P &#62; 0.05, indicating that the lack of fit was not significant. The R2 value was 0.9300, indicating that the fit of the model was good, and the error was small. It was feasible to use this model to analyze and predict the effect of the chemical composition content of the EF stir-fried with mutton oil. In addition, A2 and D2 had an effect on the content of the processed products, and the difference was statistically significant (P &#60; 0.01). The effects of A and C of the one-degree term and C2 of the second-order term on the comprehensive score were significant. The one-degree term B, the second-order A2, B2, and all of the interaction items had no significant effect on the comprehensive score. The analysis of the P values showed that, of the experimental parameters, the mutton oil amount (A) had the greatest effect on the comprehensive score, followed by the frying temperature (C), and then the mutton oil temperature (B). The above results are shown in Table 5.
The software was used to set the mutton oil amount, mutton oil temperature, and frying temperature to the medians and to use the comprehensive score as the index to draw a single-factor influence diagram of one factor (Figure 1). Increasing the frying temperature first increased the comprehensive score and then decreased it (Figure 1). The mutton oil temperature had a negligible effect on the comprehensive score. The mutton oil amount was the main significant factor that affected the change in the comprehensive score, and as the amount increased, the content trended downward.
To help better understand the results, the predicted models are presented in Figure 2 as 3D response surface plots. In terms of the slope of the response surface, the greater the significance of the interaction effect between factors, the gentler the slope, and the less significant the effect. An ellipse in the shape of a contour line indicates a strong interaction between factors, whereas a circle indicates the opposite. 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 (see Figure 2C,D), indicating that the interaction between these two factors was more significant; in contrast, the interactions between other factors were not significant (see Figure 2A,B,E,F).
The optimal mutton oil processing technology of EF was selected as follows: a mutton oil amount of 15%; a mutton oil temperature of 120 &#176;C; and a frying temperature of 189 &#176;C. Considering that the temperature cannot be very accurately controlled in actual operation, the temperature value is specified as a variable &#177;10 &#176;C. Therefore, the final parameters were as follows: a mutton oil amount of 15%; a mutton oil temperature of 120 &#176;C &#177; 10 &#176;C; and a frying temperature of 189 &#176;C &#177; 10 &#176;C. The optimal process was as follows: heating the mutton oil at 120 &#176;C &#177; 10 &#176;C, adding the crude EF, frying it with a gentle fire (189 &#176;C &#177; 10 &#176;C) until it is evenly shiny, and removing and cooling. For every 100 kg of EF, 15 kg of mutton oil (refined oil) should be used. Using these conditions, three parallel experiments were conducted, and the scores obtained were 0.96, 0.97, and 0.94 (RSD% = 1.60%), indicating stable and feasible conditions. The typical HPLC chromatograms of the crude, processed, and mixed reference substances of EF are shown in Figure 3.
Test of the effect of processing on the embryonic development of zebrafish 
The zebrafish hatched into juveniles at 72 hpf. The development of each organ was basically complete. The fish bodies remained transparent, and it was easy to lay them on their side on the glass slide. The shapes of the organs were easy to observe and identify when viewed under a microscope. The blank control group did not experience any death or organ toxicity during the administration period. Compared with the control group, at a drug concentration of 100 &#181;g/mL, no obvious abnormalities were found in the crude EF&#160;group (S) and the processed group (P) at 72 hpf. At 96 hpf and later, swim bladder incompleteness and loss of the swim bladder were more common in the juvenile fish in the crude group but were rare in the juvenile fish in the processed group. At a drug concentration of 150 &#181;g/mL, obvious spinal deformities, body curvature deformities, pericardial edema, and liver deformation were seen in the juvenile fish in the crude group at 72 hpf, but those changes were rare in the juvenile fish in the processed group, and the degree of teratogenicity was weaker than that of the crude group. At a drug concentration of 200 &#181;g/mL, all juvenile fish in the crude group died, and obvious teratogenicity appeared in the juvenile fish in the processed group. At a drug concentration of 250 &#181;g/mL, a small number of zebrafish survived in the processed group. The microscopic examination results of the zebrafish are shown in Figure 4.
The zebrafish mortality rates in the crude and processed Epimedium herb groups depended on the concentration and time of administration. The time-dose-mortality relationship is shown in Figure 5. The zebrafish mortality results showed that 24 h after administration (48 hpf), at a drug concentration of 200 &#181;g/mL, all zebrafish in the crude drug group died, whereas the mortality in the processed group was only 6.67%. At 48 h after EF administration (72 hpf), the concentration that caused the death of all zebrafish in the crude drug group was 200 &#181;g/mL, and the concentration that caused the death of all zebrafish in the processed group was 500 &#181;g/mL. The median lethal concentration of the two experimental groups at 72 hpf was calculated. The results showed that the LC50 (see Figure 6) was 151.3 &#181;g/mL in the crude group (S) and 219.8 &#181;g/mL in the processed group (P).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65096/65096fig01.jpg" /> 
Figure 1: Univariate analysis. The figure shows the single-factor influence diagram. A is the single-factor result of the amount of mutton (suet) oil; B is the single-factor result of the temperature of the mutton (suet)oil; and C is the single-factor result of the frying temperature. With an increasing frying temperature, the comprehensive score first increases and then decreases. The mutton-oil temperature has little effect on the score. The amount of mutton oil was the main significant factor affecting the change in the comprehensive score, and the content showed a downward trend with an increasing amount of mutton oil. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65096/65096fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65096/65096fig02.jpg" /> 
Figure 2: Response surface and contour plot of the influence of different factor interactions on the comprehensive score. (A) This figure shows a 3D response surface plot of the interaction between the mutton oil amount and temperature. (B) This figure shows a contour plot of the interaction between mutton oil amount and temperature. (C) This figure shows a 3D response surface plot of the interaction between the mutton oil amount and processing temperature. (D) This figure shows a contour plot of the interaction between mutton oil dosage and processing temperature. (E) This figure shows a 3D response surface plot of the interaction between mutton oil amount and processing temperature. (F) This figure shows a contour plot of the interaction between mutton oil amount and processing temperature. The result shows that the response surface of the mutton oil amount and frying temperature was steep, than the other tested parameters and the contour lines tended to be elliptical (see C,D), indicating that the interaction between these two factors was significant, whereas the interactions between other factors were not significant (see A,B,E,F). The suet oil term used in the figure refers to mutton oil. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65096/65096fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65096/65096fig03.jpg" /> 
Figure 3: HPLC chromatograms of the crude, processed, and mixed reference substances of EF. (A) This figure shows the HPLC chromatogram of the mixed reference substance. (B) This figure shows the HPLC chromatogram of crude Epimedii folium. (C) This figure shows the HPLC chromatogram of Epimedii folium processed products. These three pictures demonstrate that the BI content in raw EF is low, while it increases after processing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65096/65096fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65096/65096fig04.jpg" /> 
Figure 4: Micrographs of zebrafish. This figure shows micrographs of the zebrafish. (A) This figure shows the results for the observation of the zebrafish under a microscope in the blank group. (B) This figure shows the results for the observation of the zebrafish under a microscope in the crude group. (C) This figure shows the results for the observation of the zebrafish under a microscope in the processed group. The blank control group did not experience any death or organ toxicity during the administration period. At an EF drug concentration of 150 &#181;g/mL, obvious spinal deformities, body curvature, pericardial edema, and liver deformation were seen in the juvenile fish in the crude group at 72 hpf, whereas those changes were rare in juvenile fish in the processed group, and the degree of teratogenicity was weaker than that in the crude group. At a drug concentration of 200 &#181;g/mL, all the juvenile fish in the crude group died, and obvious teratogenicity appeared in the processed group. At a drug concentration of 250 &#181;g/mL, only a small number of zebrafish survived in the processed group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65096/65096fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65096/65096fig05.jpg" /> 
Figure 5: The dosing time-dose-mortality relationship. This figure shows the dosing time-dose-mortality relationship. (A) This figure shows the dosing time-dose-mortality relationship of the crude group. (B) This figure shows the dosing time-dose-mortality relationship of the processed group. n = 40. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65096/65096fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65096/65096fig06.jpg" /> 
Figure 6: LC50 diagram of crude and processed EF. The LC50 diagram of the crude and processed EF is shown. The median lethal concentrations of the two experimental groups at 72 hpf were calculated. The LC50 was 151.3 &#181;g/mL in the crude group (S) and 219.8 &#181;g/mL in the processing group (P). n = 40. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65096/65096fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Experimental design and the Box-Behnken response surface method results of the 17 groups of experiments. Table 1 shows the 17 groups of experiments designed by the Box-Behnken design-response surface method and their comprehensive score results. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65096/Table 1 .xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Variables used in the Box-Behnken design. The independent and dependent variables are listed here along with their low, middle, and high levels. The Box-Behnken design enabled the identification of the most influential factors in the EF processing, with the mutton oil amount (A) (15%-35%), mutton oil temperature (B) (50 &#176;C-120 &#176;C), and frying temperature (C) (80 &#176;C-300 &#176;C) as the influencing factors. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65096/Table 2 .xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Regression equations and linear ranges of the chemical constituents of EF. The results of the regression equation and linear range of the EF chemical composition show that there was good linearity between each of the concentrations of icariin, EA, EB, EC, and BI and their chromatographic peak areas. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65096/Table 3 .xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: Sample recovery test rates. The average recovery rates of icariin, EA, EB, EC, and BI were 99.98%, 100.14%, 100.09%, 100.75%, and 100.94%, respectively, and the RSD% values were 0.56%, 0.78%, 0.84%, 1.10%, and 1.47%, respectively. The results show that the accuracy of the method was suitable. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65096/Table 4 .xlsx" target="_blank">Please click here to download this Table.</a>
Table 5: Regression coefficients of the predicted quadratic model. The P value of the model was P &#60; 0.01, indicating that the model was significant. The P value of the lack of fit was P &#62; 0.05, indicating that the lack of fit was not significant. The R2 value was 0.9300, indicating that the fit of the model was good, and the error was small, so the model was suitable for analyzing and predicting the effect of the chemical composition content of the EF stir-fried with mutton oil. In addition, A2 and D2 had significant effects on the content of processed products (P &#60; 0.01). The influences of A and C of the one-degree term and C2 of the second-order term on the comprehensive score were significant. The one-degree term B, the second-order A2, B2, and all of the interaction items had no significant effects on the comprehensive score. The analysis of the P value showed that, of the experimental parameters, the amount of mutton oil (A) had the greatest influence on the comprehensive score, followed by the frying temperature (C), and then the temperature of the mutton oil (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65096/Table 5 .xlsx" target="_blank">Please click here to download this Table.</a>

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 ...

optimization-epimedii-folium-mutton-oil-processing-technology-testing

Nanyang Technological University

Research

JoVE Journal

Medicine

397 Views.  Chongqing Academy of Chinese Materia Medica. 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.

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

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg dimethylnitrosamine (DMN), given intraperitoneally for three consecutive days per week. Intraperitoneal injections were performed in the fume hood as DMN is a known hepatoxin and carcinogen. The model has several advantages. Firstly, liver changes can be studied sequentially or at particular stages of interest. Secondly, the stage of liver disease can be monitored by measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. Thirdly, the severity of liver damage at different stages can be confirmed by sacrifice of animals at designated time points, followed by histological examination of Masson&#39;s Trichome stained liver tissues. After four weeks of DMN dosing, the typical fibrosis score is 5 to 6 on the Ishak scale. The model can be reproduced consistently and has been widely used to assess the efficacy of potential anti-fibrotic agents.

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg ...

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

A Quantitative Sensory Testing Paradigm to Obtain Measures of Pain Processing in Patients Undergoing Breast Cancer Surgery

Chronic pain following surgery, persistent postsurgical pain, is an important highly prevalent condition contributing to significant symptom burden and lower quality of life. Persistent postsurgical pain is relatively refractory to treatment hence generating a high need for preventive strategies and treatments. Therefore, the identification of patients at risk of developing persistent pain is an area of active ongoing research. Recently it was demonstrated that peri-operative disruptions in central pain processing may be able to predict persistent postsurgical pain at long term follow-up in breast cancer patients. The aim of the current report is to present a short protocol to obtain pain thresholds to different stimuli at multiple sites and a measure of endogenous analgesia in breast cancer patients. We have used this method successfully in a clinical context and detail some representative results from a clinical study.

Persistent postsurgical pain may be related to changes in pain processing. By assessing pain in response to standardized stimuli, changes in pain processing can be elucidated. We present methods to obtain pain thresholds to different stimuli and a measure of endogenous analgesia in patients undergoing breast cancer surgery.

Chronic pain following surgery, persistent postsurgical pain, is an important highly prevalent condition contributing to significant symptom burden and ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Development and Implementation of a Multi-Disciplinary Technology Enhanced Care Pathway for Youth and Adults with Concussion

The evidence-informed standardization of care along disease lines is recommended to improve outcomes and reduce healthcare costs. The aim of this project is to 1) describe the development and implementation of the Concussion Carepath, 2) demonstrate the process of integrating technology in the form of a mobile application to enable the carepath and guide clinical decision-making, and 3) present data on the utility of the C3 app in facilitating decision-making throughout the injury recovery process. A multi-disciplinary team of experts in concussion care was formed to develop an evidence-informed algorithm, outlining best practices for the clinical management of concussion along three phases of recovery &#8211; acute, subacute, and post-concussive. A custom mobile application, the Cleveland Clinic Concussion (C3) app was developed and validated to provide a platform for the systematic collection of objective, biomechanical outcomes and to provide guidance in clinical decision-making in the field and clinical environments. The Cleveland Clinic Concussion app included an electronic incident report, assessment modules to measure important aspects of cognitive and motor function, and a return to play module to systematically document the six phases of post-injury rehabilitation. The assessment modules served as qualifiers within the carepath algorithm, driving referral for specialty services as indicated. Overall, the carepath coupled with the C3 app functioned in unison to facilitate communication among the interdisciplinary team, prevent stagnant care, and drive patients to the right provider at the right time for efficient and effective clinical management.

A multidisciplinary carepath to standardize disparate concussion care was developed and implemented at the Cleveland Clinic. The Cleveland Clinic Concussion (C3) mobile application was used to enable the carepath through biomechanical outcomes characterizing cognitive and motor function. Patient outcomes improved while cost of care was reduced following implementation.

The evidence-informed standardization of care along disease lines is recommended to improve outcomes and reduce healthcare costs. The aim of this project ...