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

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

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

<ol>
	<li>Chinese Pharmacopoeia Commission. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chinese Pharmacopoeia. Volume I. , (2020).</li><li>Wang, X. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Collection of Traditional Chinese Medicine Processing Methods. , (1998).</li><li>Chen, L. L., Jia, X. B., Jia, D. S. <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+studies+on+processing+mechanism+of+Epimedii+Folium.">Advances in studies on processing mechanism of Epimedii Folium.</a> Chinese Traditional and Herbal Drugs. 12 (12), 2108-2111 (2010).</li><li>Zhao, W., 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=Optimized+extraction+of+polysaccharides+from+corn+silk+by+pulsed+electric+field+and+response+surface+quadratic+design.">Optimized extraction of polysaccharides from corn silk by pulsed electric field and response surface quadratic design.</a> Journal of The Science of Food and Agriculture. 91 (12), 2201-2209 (2011).</li><li>Zhao, L. C., 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=The+use+of+response+surface+methodology+to+optimize+the+ultrasound-assisted+extraction+of+five+anthraquinones+from+Rheum+palmatum+L.">The use of response surface methodology to optimize the ultrasound-assisted extraction of five anthraquinones from Rheum palmatum L.</a> Molecules. 16 (7), 5928-5937 (2011).</li><li>Mao, W. H., Han, L. J., Shi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+microwave+assisted+extraction+of+flavonoid+from+Radix+Astragali+using+response+surface+methodology.">Optimization of microwave assisted extraction of flavonoid from Radix Astragali using response surface methodology.</a> Separation Science and Technology. 43 (12), 671-681 (2008).</li><li>Liu, W., 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=Optimization+of+total+flavonoid+compound+extraction+from+Gynura+medica+leaf+using+response+surface+methodology+and+chemical+composition+analysis.">Optimization of total flavonoid compound extraction from Gynura medica leaf using response surface methodology and chemical composition analysis.</a> International Journal of Molecular Sciences. 11 (11), 4750-4763 (2010).</li><li>Guo, G. 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=Research+progress+on+processing+mechanism+of+Epimedium+fried+with+sheep+fat+oil+based+on+warming+kidney+and+promoting+yang.">Research progress on processing mechanism of Epimedium fried with sheep fat oil based on warming kidney and promoting yang.</a> Journal of Liaoning University of TCM. 22 (07), 1-5 (2020).</li><li>Shen, X. J., Zhou, Q., Sun, L. -. L., Dai, Y. -. P., Yan, X. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+for+cutting+procedure+of+astragali+radix+with+Box-Behnken+design+and+response+surface+method.">Optimization for cutting procedure of astragali radix with Box-Behnken design and response surface method.</a> China Journal of Chinese Materia Medica. 39 (13), 2498-2503 (2014).</li><li>Wang, L. H., 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=Optimization+of+processing+technology+of+honey+wheat+bran+based+on+Box-Behnken+response+surface+methodology.">Optimization of processing technology of honey wheat bran based on Box-Behnken response surface methodology.</a> Chinese Traditional and Herbal Drugs. 52 (12), 3538-3543 (2021).</li><li>Zhang, J. B., 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=Study+on+integrated+process+of+producing+area+and+processing+production+for+Paeoniae+Radix+Alba+based+on+Box-Behnken+response+surface+methodology.">Study on integrated process of producing area and processing production for Paeoniae Radix Alba based on Box-Behnken response surface methodology.</a> Chinese Traditional and Herbal Drugs. 53 (18), 5657-5662 (2022).</li><li>Li, N., Zhang, X. M., Yao, Y. Y., Chen, Y. L., Fan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+processing+technology+for+Psoraleae+Fructus+by+D-optimal+response+surface+methodology+with+UHPLC.">Optimization of processing technology for Psoraleae Fructus by D-optimal response surface methodology with UHPLC.</a> Chinese Traditional and Herbal Drugs. 39 (05), 42-44 (2022).</li><li>Jia, Y. Q., 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=Optimization+of+processing+technology+with+wine+of+Cnidii+Fructus+by+AHP-entropy+weight+method+combined+with+response+surface+method.">Optimization of processing technology with wine of Cnidii Fructus by AHP-entropy weight method combined with response surface method.</a> Journal of Chinese Medicinal Materials. 10, 2338-2343 (2022).</li><li>Chen, F. G., 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=Optimization+of+the+baked+drying+technology+of+Cinnamomi+Ramulus+based+on+CRITIC+combined+with+Box-Behnken+response+surface+method.">Optimization of the baked drying technology of Cinnamomi Ramulus based on CRITIC combined with Box-Behnken response surface method.</a> Journal of Chinese Medicinal Materials. 2022 (08), 1838-1842 (2022).</li><li>Wang, W. D., 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=Optimization+extraction+of+effective+constituents+from+Epimedii+Herba+based+on+central+composite+design-response+surface+methodology+and+orthogonal+experimental+design.">Optimization extraction of effective constituents from Epimedii Herba based on central composite design-response surface methodology and orthogonal experimental design.</a> Lishizhen Medicine and Materia Medica. 21 (11), 2766-2768 (2010).</li><li>Yang, 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=Zebrafish+embryos+as+models+for+embryotoxic+and+teratological+effects+of+chemicals.">Zebrafish embryos as models for embryotoxic and teratological effects of chemicals.</a> Reproductive Toxicology. 28 (2), 245-253 (2009).</li><li>Kanungo, J., Cuevas, E., Ali, S. F., Paule, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+model+in+drug+safety+assessment.">Zebrafish model in drug safety assessment.</a> Current Pharmaceutical Design. 20 (34), 5416-5429 (2014).</li><li>Jayasinghe, C. D., Jayawardena, U. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+assessment+of+herbal+medicine+using+zebrafish+embryos:+A+systematic+review.">Toxicity assessment of herbal medicine using zebrafish embryos: A systematic review.</a> Evidence-Based Complementary and Alternative Medicine. 2019, 7272808 (2019).</li><li>Scholz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+embryos+as+an+alternative+model+for+screening+of+drug+induced+organ+toxicity.">Zebrafish embryos as an alternative model for screening of drug induced organ toxicity.</a> Archives of Toxicology. 87 (5), 767-769 (2013).</li><li>Ling, J., 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=Analysis+of+Folium+Epimedium+toxicity+in+combination+with+Radix+Morindae+Officinalis+based+on+zebrafish+toxicity/metabolism+synchronization.">Analysis of Folium Epimedium toxicity in combination with Radix Morindae Officinalis based on zebrafish toxicity/metabolism synchronization.</a> Acta Pharmaceutica Sinica. 53 (1), 74 (2018).</li><li>Wang, 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=Tri-n-butyl+phosphate+delays+tissue+repair+by+dysregulating+neutrophil+function+in+zebrafish.">Tri-n-butyl phosphate delays tissue repair by dysregulating neutrophil function in zebrafish.</a> Toxicology and Applied Pharmacology. 449, 116114 (2022).</li><li>Sheng, Z. L., Li, J. C., Li, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+forsythoside+extraction+from+Forsythia+suspensa+by+Box-Behnken+design.">Optimization of forsythoside extraction from Forsythia suspensa by Box-Behnken design.</a> African Journal of Biotechnology. 10 (55), 11728-11737 (2011).</li><li>Pang, X., 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=Prenylated+flavonoids+and+dihydrophenanthrenes+from+the+leaves+of+Epimedium+brevicornu+and+their+cytotoxicity+against+HepG2+cells.">Prenylated flavonoids and dihydrophenanthrenes from the leaves of Epimedium brevicornu and their cytotoxicity against HepG2 cells.</a> Natural Product Research. 32 (19), 2253-2259 (2018).</li><li>Zhong, R., 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=The+toxicity+and+metabolism+properties+of+Herba+Epimedii+flavonoids+on+laval+and+adult+zebrafish.">The toxicity and metabolism properties of Herba Epimedii flavonoids on laval and adult zebrafish.</a> Evidence-Based Complementary and Alternative Medicine. 2019, 3745051 (2019).</li><li>Zhang, 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=Effect+of+2&amp;#34;+-O-rhamnosyl+icariside+II,+baohuoside+I+and+baohuoside+II+in+Herba+Epimedii+on+cytotoxicity+indices+in+HL-7702+and+HepG2+cells.">Effect of 2&amp;#34; -O-rhamnosyl icariside II, baohuoside I and baohuoside II in Herba Epimedii on cytotoxicity indices in HL-7702 and HepG2 cells.</a> Molecules. 24 (7), 1263 (2019).</li><li>Chen, Y., Yang, R. J., Yu, M., Ding, S. L., Chen, R. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+response+surface+methodology+in+modern+production+process+optimization.">Application of response surface methodology in modern production process optimization.</a> Science &amp; Technology Vision. 2016 (19), 36-39 (2016).</li><li>Zhang, 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=Progress+in+using+zebrafish+as+a+toxicological+model+for+traditional+Chinese+medicine.">Progress in using zebrafish as a toxicological model for traditional Chinese medicine.</a> Journal of Ethnopharmacology. 282, 114638 (2022).</li><li>Oliveira, R., Domingues, I., Grisolia, C. K., Soares, A. M. V. M. <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+triclosan+on+zebrafish+early-life+stages+and+adults.">Effects of triclosan on zebrafish early-life stages and adults.</a> Environmental Science and Pollution Research. 16 (6), 679-688 (2009).</li><li>Ton, C., Lin, Y., Willett, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+for+developmental+neurotoxicity+testing.">Zebrafish as a model for developmental neurotoxicity testing.</a> Birth Defects Research. Part A, Clinical and Molecular Teratology. 76 (7), 553-567 (2006).</li><li>He, Q., 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=Toxicity+induced+by+emodin+on+zebrafish+embryos.">Toxicity induced by emodin on zebrafish embryos.</a> Drug and Chemical Toxicology. 35 (2), 149-154 (2012).</li><li>Chen, 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=Developmental+toxicity+of+muscone+on+zebrafish+embryos.">Developmental toxicity of muscone on zebrafish embryos.</a> Chinese Journal of Pharmacology and Toxicology. (6), 267-273 (2014).</li><li>He, 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+shikonin+on+zebrafish's+embryo+and+angiogenesis.">Effects of shikonin on zebrafish's embryo and angiogenesis.</a> Chinese Traditional Patent Medicine. 38 (2), 241-245 (2016).</li><li>Zhou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The transformation research on the chemical compositions in the processing of Epimedium. , (2016).</li><li>Xiao, Y. P., Zeng, J., Jiao, L. -. N., Xu, X. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+for+treatment+effect+and+signaling+pathway+regulation+of+kidney-tonifying+traditional+Chinese+medicine+on+osteoporosis.">Review for treatment effect and signaling pathway regulation of kidney-tonifying traditional Chinese medicine on osteoporosis.</a> China Journal of Chinese Materia Medica. 43 (1), 21-30 (2018).</li><li>Wang, R. 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>

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

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

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

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

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

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

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

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

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

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

In this study, we adopted a widely used processing method and acquired a stable and reliable persistent technique. The result showed that the toxicity of processed Epimedii was reduced. Response surface method requires fewer experiments and shorter cycles, which cannot only avoid the problems existing in traditional mathematical statistic, but also help users to select the optimal conditions.This method can also be widely used in the chemical industry, biologic engineering, food industry, Chinese medicine preparation, Chinese medicine extraction, and so on. After preparing the solution and performing the chromatography, select the key parameters in EF processing, such as the amount of mutton oil, the mutton oil temperature, and the frying temperature as influential factors. Use the comprehensive scores of icariin, EA, EB, EC, and BI content as the evaluation indexes.Use the response surface analysis software to design the Box-Behnken response surface experiments, explore the quadratic response surface, and construct a second order polynomial model. Select the new Box-Behnken design, and set the numeric factors option to three. Set factors A, B, and C.Click on continue, then set the responses option to one, and click on continue to complete the design.Process the EF according to the specific parameters, for example, for order number one, where refined mutton oil as 15%volume by volume, and then heat it to 50 degrees Celsius to melt it. Add the crude EF to the melted mutton. Stir fry over a gentle fire until it is evenly shiny, then remove and cool the EF.Prepare the test solutions of the processed products, and analyze them using HPLC as described in the manuscript.Record the retention times and peak areas of each chemical composition, and calculate the contents of the icariin, EA, EB, EC, and BI in each test solution against an external standard curve. Calculate the comprehensive scores of the 17 experimental groups using the comprehensive score calculation formula. Import the comprehensive scoring results for the 17 groups of experiments into the data analysis software to analyze the experimental data.Under the evaluation items, select the quadratic process order option and polynomial model type option. The response surface of the mutton oil amount and the frying temperature was steeper compared to the other tested factors, and the contour lines tended to be more elliptical, indicating that the interaction between these two factors was more significant than the interactions between other factors. The HPLC chromatogram of the mixed reference substance, crude Epimedii Folium and Epimedii Folium processed products demonstrate that the BI content in raw EF is low, while it increases after processing.The crude group shows obvious spinal deformities, body curvature, pericardial edema, and liver deformation at 150 micrograms per milliliter drug concentration, whereas those changes were rare in juvenile fish in the processed group. All zebrafish in the crude drug group died 24 hours after 200 micrograms per milliliter drug administration, whereas the mortality in the processed group was only 6.67%Administration of 200 micrograms per milliliter of EF caused the death of all zebrafish in the crude drug group after 72 hours, while in the processed group, 500 micrograms per milliliter of EF caused the death of all zebrafish. The median lethal concentration of the two experimental groups at 72 hours post fertilization showed that the LC 50 was 151.3 micrograms per milliliter in the crude group, and 219.8 micrograms per milliliter in the processed group.The most important thing in the experiment is selecting factors of the response surface method, and determine the parameters of these factors.

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

Research

JoVE Journal

Medicine

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 ...

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 ...

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 ...

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...

A Method for Zanba-Stir-Fried Tiebangchui Process Optimization

Optimization of Processing Technology for Tiebangchui with Zanba Based on CRITIC Combined with Box-Behnken Response Surface Method

The dried root of Aconitum pendulum Busch., called Tiebangchui (TBC) in Chinese, is one of the most famous Tibetan medicines. It is a widely used herb in northwest China. However, many cases of poisoning have occurred because of TBC's intense toxicity and because its therapeutic and toxic doses are similar. Therefore, finding a safe and effective method to reduce its toxicity is an urgent task. A search through the Tibetan medicine classics shows that the processing method of TBC stir-fried with Zanba was recorded in the "Processing specification of Tibetan medicine of Qinghai Province (2010)". However, the specific processing parameters are not yet clear. Thus, this study aims to optimize and standardize the processing technology of Zanba-stir-fried TBC.
First, a single-factor experiment was conducted on four factors: the slice thickness of TBC, amount of Zanba, processing temperature, and time. With monoester and diester alkaloid contents in Zanba-stir-fried TBC as indexes, CRITIC combined with the Box-Behnken response surface method was used to optimize the processing technology of Zanba-stir-fried TBC. The optimized processing conditions of Zanba-stir-fried TBC were a TBC slice thickness of 2 cm, three times more Zanba than TBC, a processing temperature of 125 &#176;C, and 60 min of stir-frying. This study determined the optimized and standard processing conditions for the usage of Zanba-stir-fried TBC, thus providing an experimental basis for the safe clinical use and industrial production of Zanba-stir-fried TBC.

Author Spotlight: Optimization of Processing Technology for Tiebangchui with Zanba Based on CRITIC Combined with Box-Behnken Response Surface Method  

The present protocol describes an efficient and standard detoxification processing method for Zanba-stir-fried Tiebangchui using CRITIC combined with the Box-Behnken response surface method.

The dried root of Aconitum pendulum Busch., called Tiebangchui (TBC) in Chinese, is one of the most famous Tibetan medicines. It is a widely used herb in ...

Optimization of Processing of Tiebangchui with Highland Barley Wine Based on the Box-Behnken Design Combined with the Entropy Method

The processing of toxic ethnomedicines is of great significance for their safe clinical application. Thus, the limitations of traditional processing should be addressed, and the processing method of ethnomedicines should be standardized using modern research methods. In this study, the processing technology of a commonly used Tibetan medicine Tiebangchui (TBC), the dried root of Aconitum pendulum&#160;Busch, processed with highland barley wine was optimized. Diester-diterpenoid alkaloid (DDA) (aconitine, 3-deoxyaconitine, 3-acetylaconitine) and monoester-diterpenoid alkaloid (MDA) (benzoylaconine) content were used as evaluation indicators, and the weight coefficient of each evaluation index was determined by the entropy method.
The single factor test and Box-Behnken design were used in investigating the influence of the ratio between highland barley wine and TBC, slice thickness of TBC, and processing time. Comprehensive scoring was performed according to the objective weight of each index determined by the entropy method. The optimal processing conditions of TBC with highland barley wine were as follows: the amount of highland barley wine is five times that of TBC, a soaking time of 24 h, and a TBC thickness of 1.5 cm. The results showed that the relative standard deviation between the verification test and predicted value was less than 2.55% and the optimized processing technology of TBC processed with highland barley wine is simple, feasible, and stable, and so can provide a reference for industrial production.

The present protocol describes an efficient method for optimization of the processing technology of Tiebangchui processed with highland barley wine based on a Box-Behnken design response surface combined with the entropy method.

The processing of toxic ethnomedicines is of great significance for their safe clinical application. Thus, the limitations of traditional processing ...

Preparing Zanba-Stir-Fried Tiebangchui (TBC) Sample for Processing Technology Optimization

To begin a single factor experiment to compare Tiebangchui, or TBC slice thickness, for processing technology optimization, prepare five test groups each having 30 grams of TBC, but different slice thickness. Then weigh 90 grams of Zanba, which is three times as much as that of the TBC in each group. Set the temperature and the time of the automatic stir fry machine to 140 degrees Celsius and 40 minutes respectively.After the machine has heated up to the set temperature, add the weighed amounts of TBC and Zanba into the machine. Then using a high speed smashing machine, grind the Zanba stir-fried TBC into powder. Next, weigh two grams of Zanba stir-fried TBC powder in a conical flask.To the sample in the flask, add three milliliters of ammonia solution followed by 50 milliliters of a mixture of isopropyl alcohol and ethyl acetate, having a volume ratio of one is to one. After recording the weight of the conical flask with the contents, ultrasonicate the TBC sample for 30 minutes at 220 volts and 40 kilohertz maintaining a temperature below 25 degrees Celsius. Weigh the conical flask with the sample once again after the ultrasonic extraction.Make up for the lost weight by adding the isopropyl alcohol and ethyl acetate mixture mentioned previously, and filter the sample solution. Evaporate 25 milliliters of the filtrate to dryness using a rotary evaporator at 40 degrees Celsius. Then dissolve the residue in five milliliters of a solution of 0.05 hydrochloric acid in methanol.Filter the solution through a 0.2 micron syringe filter, and analyze it by performing high performance liquid chromatography, or HPLC. Calculate the contents of the MDA and DDAs in different processing products according to the standard curve. Calculate the comprehensive score based on the results via the critic method.Following the method demonstrated above, compare the amounts of Zanba, processing temperatures, and times for processing technology optimization. The results of the single factor tests revealed that the comprehensive scores of Zanba stir-fried TBC were highest when the sliced thickness is two centimeters, the amount of Zanba is three times, the processing temperature is 120 degrees Celsius, and the processing time is 60 minutes. The results revealed the range of slicing thickness, amount of Zanba, processing temperature, and processing time to be used for future experiments.