JoVE Journal > Medicine
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Declarações
Acknowledgements
Materials
Referências
Tadução automática
Please note that all translations are automatically generated. Click here for the English version.
Medicina

淫羊藿叶羊油加工技术优化及检验其对斑马鱼胚胎发育的影响

Published: March 17, 2023
doi:
10.3791/65096
, , , , , ,
1Chongqing Academy of Chinese Materia Medica, 2Bioengineering College of Chongqing University

Summary

本实验采用Box-Behnken实验设计-响应面法优化了羊藿果油加工技术，初步研究了粗提和优化水提取EF对斑马鱼胚胎发育的影响。

Abstract

作为一种传统中药（TCM），淫羊藿（EF）在医学和食品方面已有>2000年的历史。临床上，用羊油加工的EF常被用作药物。近年来，使用EF作为原料的产品的安全风险和不良反应的报告逐渐增加。加工可以有效提高中药的安全性。根据中医理论，羊油加工可以降低EF的毒性，增强其对肾脏的滋补作用。然而，对EF羊油加工技术缺乏系统的研究和评价。本研究采用Box-Behnken实验设计-响应曲面方法，通过评估多个组分的含量来优化加工技术的关键参数。结果表明，EF的最佳羊油加工工艺为：将羊油在120 °C±10 °C加热，加入粗EF，在189 °C±10 °C轻轻翻炒至均匀光泽，然后取出冷却。每100公斤EF，应使用15公斤羊油。在斑马鱼胚胎发育模型中比较了粗水和羊油加工EF水提取物的毒性和致畸性。结果表明，粗草本组更容易引起斑马鱼畸形，其半最大致死EF浓度较低。综上所述，优化后的羊油加工技术稳定可靠，重复性好。在一定剂量下，EF水提取物对斑马鱼胚胎的发育有毒，对生药的毒性强于对加工药物的毒性。结果表明，羊油加工降低了原油EF的毒性。这些发现可用于提高羊油加工EF的质量、均匀性和临床安全性。

Introduction

Epimedii folium（EF）是Epimedium brevicornu Maxim.，Epimedium sagittatum （Sieb. et Zucc.）的干燥叶子。Maxim.，Epimedium pubescens Maxim.，或Epimedium Koreanum Nakai。EF可用于治疗骨质疏松症、更年期综合征、乳腺肿块、高血压、冠心病等疾病1.作为一种传统中药（TCM），EF在医学和食品领域已有2000多年的历史。由于其价格低廉，补肾效果显著，被广泛用于医药和保健食品。EF是用羊油炒制而成的，这一过程最早在刘宋时期雷霄撰写的《雷功加工理论》中有所描述。粗EF和炒EF的功效完全不同。粗EF主要祛风湿，而炒EF则温肾补阳3。目前，EF被广泛用作药品和保健食品的原料;上市中成药399种，进口保健食品9种，以EF为原料的国产保健食品455种。该药材具有很大的应用前景。但近年来，保健食品和中成药以EF为原料引起不良反应和人体肝损伤的报道越来越多，相关毒性研究5、6、7报道EF作为原料存在安全隐患。

中药加工是指能够有效降低或消除毒性，提高中药安全性的制药技术。EF的传统加工方法是用羊油炒，降低了EF的毒性，增强了其温肾益阳的作用8。此加工方法收录于《中国药典》及各种加工规范1.EF的工艺仅规定如下：每100公斤EF加入20公斤羊油（精制），温和烧制至均匀有光泽1.上述标准中没有严格的EF处理方法参数，因此当地的处理规范尚未统一以提供一致性。因此，对EF过程进行系统研究将是有用的。本文采用Box-Behnken实验设计-响应面方法对EF的加工工艺进行了优化。

Box-Behnken 实验设计是一种通常用于优化过程中因子的方法。通过建立多元回归方程拟合因子与效应值之间的函数关系，可以优化提取参数。最近，该方法已被广泛用于研究中药提取5，6，7和加工9，10，11。各种研究报道了中药制备方法，包括盐加工、葡萄酒加工和按照Box-Behnken设计进行炒菜，例如盐加工补骨脂12，葡萄酒加工的刺胞菌13和烤肉桂14。该方法测试时间短，测试精度高，适用于多因素、多级测试。该方法比正交设计试验方法简单，比统一设计方法15更全面。获得的关系可以确定测试范围内任何测试点的预测值，这是一个很大的优势。斑马鱼模型可用于测试EF在加工后的毒性是否较低。

在中药毒性研究中，斑马鱼模型具有细胞实验通量高和与啮齿动物实验相似性的双重优势16。该模型的特点是体积小，产卵率高，繁殖周期短，易于繁殖。该模型可用于细胞培养板的大规模同步实验，实验用药量小，实验周期短，成本低，整个实验过程易于观察和操作17.斑马鱼胚胎是透明的，发育迅速。因此，可以在显微镜下直接观察到药物在不同发育阶段对内脏组织的毒性和致畸作用18。斑马鱼与人类的基因同源性高达85%18。斑马鱼的信号转导途径与人类相似18。斑马鱼的生物结构和生理功能与哺乳动物高度相似18。因此，用于药物测试的斑马鱼模型可以提供可靠且完全适用于人类的实验动物19。

本研究采用Box-Behnken设计-响应面法，以淫羊藿苷、淫羊藿素A、淫羊藿素B、淫羊藿素C、宝贝壳苷I等含量为评价指标，对EF加工工艺中羊肉油的用量、温度和油炸温度进行优化。利用斑马鱼模型初步探讨EF水提取物对斑马鱼加工前后胚胎发育的影响，评价加工对EF的衰减效果。

Protocol

所有动物相关实验均经重庆中医药研究院实验伦理委员会批准进行（实验动物伦理审查证书编号：ZJS2022-03）。 1. 生物活性成分的测定 注：本研究使用的物种为 淫羊藿矢状，样本采集于重庆丰都县。该样本被鉴定为 E. sagittatum （Sieb. et Zucc.）的干燥地上部分。格言。由重庆中医药研究所生物医学研究所的研究人员提供。 ?…

Representative Results

方法学调查结果观察到淫羊藿苷、EA、EB、EC、BI的浓度与色谱峰面积之间存在线性关系（见 表3）。淫羊藿苷、EA、EB、EC和BI色谱峰面积的RSD%值（n = 6）分别为0.28%、1.22%、0.65%、1.67%和1.06%，表明HPLC测量的精密度良好。淫羊藿苷、EA、EB、EC和BI含量的RSD%值（n=6）分别为1.59%、1.46%、1.86%、2.29%和0.98%，表明该方法具有良好的重复性。样品中淫羊藿苷、EA、EB、EC和BI峰面积的RSD…

Discussion

自变量及其水平的确定
EF加工技术仅在2020年版《中国药典》和全国26个省、市、自治区公布的地方中药加工规范中有所描述1。描述涉及以下步骤：取羊油加热融化，加入EF丝，用慢火炒至均匀有光泽，取出，冷却。此外，每 100 公斤淫羊藿使用 20 公斤（缩写为 20%）羊油（精制）。但是，未指定EF处理过程的参数。本实验的自变量中，可以量化生产过程中的三个关?…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作得到了重庆市中医药研究院基础科研业务项目（项目编号：jbky20200013）、重庆市科研机构绩效激励引导项目（项目编号：cstc2021jxjl 130025）和重庆市卫生健康委中药材加工重点学科建设项目的支持。

Materials

Acetonitrile Fisher 197164
Baohuoside Equation 1 (BEquation 1 )  Chengdu Manst Biotechnology Co., Ltd. MUST-20042402
Chromatographic column Waters Corporation Symmetry C18
Design Expert software Stat- Ease Inc., Minneapolis, MN Trial Version8.0.6.1
Detector Waters Corporation 2998
Disintegrator Hefei Rongshida Small Household Appliance Co., Ltd. S-FS553
Electronic analytical balance Mettler-Toledo International Inc. MS205DU
Epimedin A (EA) Chengdu Manst Biotechnology Co., Ltd. MUST-21112118
Epimedin B (EB) Chengdu Manst Biotechnology Co., Ltd. MUST-20080403
Epimedin C (EC) Chengdu Manst Biotechnology Co., Ltd. MUST-20080310
Ethanol Chongqing Chuandong Chemical ( Group ) Co., Ltd. 20180801
Graphpad software GraphPad Software Inc., San Diego, CA, USA 6.02
High Performance Liquid Chromatography (HPLC) Waters Corporation 2695
Icariin Chengdu Glip Biotechnology Co., Ltd. 21091401
Methanol Chongqing Chuandong Chemical (Group) Co., Ltd. 20171101
Microporous membrane Tianjin Jinteng Experimental Equipment Co., Ltd. 0.22μm
Mutton oil Kuoshan Zhiniu Fresh Food Store 20211106
Office Excel office software Microsoft Office Excel 2021
Pharmacopoeia sieve Shaoxing Shangyu Huafeng Hardware Instrument Co., Ltd. R40/3
Pure water machine Chongqing Andersen Environmental Protection Equipment Co., Ltd. AT Sro 10A
Qualitative filter paper Shanghai Leigu Instrument Co., Ltd. 18cm
Stereomicroscope Carl Zeiss, Oberkochen, Germany Stemi 2000
Ultrasonic cleaner Branson Ultrasonics (Shanghai) Co.,Ltd. BUG25-12
Zebrafish China Zebrafish Resource Center (CZRC) The AB strain
DOWNLOAD MATERIALS LIST

Referências

  1. Chinese Pharmacopoeia Commission. . Chinese Pharmacopoeia. Volume I. , (2020).
  2. Wang, X. T. . Collection of Traditional Chinese Medicine Processing Methods. , (1998).
  3. Chen, L. L., Jia, X. B., Jia, D. S. Advances in studies on processing mechanism of Epimedii Folium. Chinese Traditional and Herbal Drugs. 12 (12), 2108-2111 (2010).
  4. Zhao, W., et al. Optimized extraction of polysaccharides from corn silk by pulsed electric field and response surface quadratic design. Journal of The Science of Food and Agriculture. 91 (12), 2201-2209 (2011).
  5. Zhao, L. C., et al. The use of response surface methodology to optimize the ultrasound-assisted extraction of five anthraquinones from Rheum palmatum L. Molecules. 16 (7), 5928-5937 (2011).
  6. Mao, W. H., Han, L. J., Shi, B. Optimization of microwave assisted extraction of flavonoid from Radix Astragali using response surface methodology. Separation Science and Technology. 43 (12), 671-681 (2008).
  7. Liu, W., et al. Optimization of total flavonoid compound extraction from Gynura medica leaf using response surface methodology and chemical composition analysis. International Journal of Molecular Sciences. 11 (11), 4750-4763 (2010).
  8. Guo, G. L., et al. Research progress on processing mechanism of Epimedium fried with sheep fat oil based on warming kidney and promoting yang. Journal of Liaoning University of TCM. 22 (07), 1-5 (2020).
  9. Shen, X. J., Zhou, Q., Sun, L. -. L., Dai, Y. -. P., Yan, X. -. S. Optimization for cutting procedure of astragali radix with Box-Behnken design and response surface method. China Journal of Chinese Materia Medica. 39 (13), 2498-2503 (2014).
  10. Wang, L. H., et al. Optimization of processing technology of honey wheat bran based on Box-Behnken response surface methodology. Chinese Traditional and Herbal Drugs. 52 (12), 3538-3543 (2021).
  11. Zhang, J. B., et al. Study on integrated process of producing area and processing production for Paeoniae Radix Alba based on Box-Behnken response surface methodology. Chinese Traditional and Herbal Drugs. 53 (18), 5657-5662 (2022).
  12. Li, N., Zhang, X. M., Yao, Y. Y., Chen, Y. L., Fan, Q. Optimization of processing technology for Psoraleae Fructus by D-optimal response surface methodology with UHPLC. Chinese Traditional and Herbal Drugs. 39 (05), 42-44 (2022).
  13. Jia, Y. Q., et al. Optimization of processing technology with wine of Cnidii Fructus by AHP-entropy weight method combined with response surface method. Journal of Chinese Medicinal Materials. 10, 2338-2343 (2022).
  14. Chen, F. G., et al. Optimization of the baked drying technology of Cinnamomi Ramulus based on CRITIC combined with Box-Behnken response surface method. Journal of Chinese Medicinal Materials. 2022 (08), 1838-1842 (2022).
  15. Wang, W. D., et al. Optimization extraction of effective constituents from Epimedii Herba based on central composite design-response surface methodology and orthogonal experimental design. Lishizhen Medicine and Materia Medica. 21 (11), 2766-2768 (2010).
  16. Yang, L., et al. Zebrafish embryos as models for embryotoxic and teratological effects of chemicals. Reproductive Toxicology. 28 (2), 245-253 (2009).
  17. Kanungo, J., Cuevas, E., Ali, S. F., Paule, M. G. Zebrafish model in drug safety assessment. Current Pharmaceutical Design. 20 (34), 5416-5429 (2014).
  18. Jayasinghe, C. D., Jayawardena, U. A. Toxicity assessment of herbal medicine using zebrafish embryos: A systematic review. Evidence-Based Complementary and Alternative Medicine. 2019, 7272808 (2019).
  19. Scholz, S. Zebrafish embryos as an alternative model for screening of drug induced organ toxicity. Archives of Toxicology. 87 (5), 767-769 (2013).
  20. Ling, J., et al. Analysis of Folium Epimedium toxicity in combination with Radix Morindae Officinalis based on zebrafish toxicity/metabolism synchronization. Acta Pharmaceutica Sinica. 53 (1), 74 (2018).
  21. Wang, Y., et al. Tri-n-butyl phosphate delays tissue repair by dysregulating neutrophil function in zebrafish. Toxicology and Applied Pharmacology. 449, 116114 (2022).
  22. Sheng, Z. L., Li, J. C., Li, Y. H. Optimization of forsythoside extraction from Forsythia suspensa by Box-Behnken design. African Journal of Biotechnology. 10 (55), 11728-11737 (2011).
  23. Pang, X., et al. Prenylated flavonoids and dihydrophenanthrenes from the leaves of Epimedium brevicornu and their cytotoxicity against HepG2 cells. Natural Product Research. 32 (19), 2253-2259 (2018).
  24. Zhong, R., et al. The toxicity and metabolism properties of Herba Epimedii flavonoids on laval and adult zebrafish. Evidence-Based Complementary and Alternative Medicine. 2019, 3745051 (2019).
  25. Zhang, L., et al. Effect of 2" -O-rhamnosyl icariside II, baohuoside I and baohuoside II in Herba Epimedii on cytotoxicity indices in HL-7702 and HepG2 cells. Molecules. 24 (7), 1263 (2019).
  26. Chen, Y., Yang, R. J., Yu, M., Ding, S. L., Chen, R. Q. Application of response surface methodology in modern production process optimization. Science & Technology Vision. 2016 (19), 36-39 (2016).
  27. Zhang, Y., et al. Progress in using zebrafish as a toxicological model for traditional Chinese medicine. Journal of Ethnopharmacology. 282, 114638 (2022).
  28. Oliveira, R., Domingues, I., Grisolia, C. K., Soares, A. M. V. M. Effects of triclosan on zebrafish early-life stages and adults. Environmental Science and Pollution Research. 16 (6), 679-688 (2009).
  29. Ton, C., Lin, Y., Willett, C. Zebrafish as a model for developmental neurotoxicity testing. Birth Defects Research. Part A, Clinical and Molecular Teratology. 76 (7), 553-567 (2006).
  30. He, Q., et al. Toxicity induced by emodin on zebrafish embryos. Drug and Chemical Toxicology. 35 (2), 149-154 (2012).
  31. Chen, Y., et al. Developmental toxicity of muscone on zebrafish embryos. Chinese Journal of Pharmacology and Toxicology. (6), 267-273 (2014).
  32. He, Y. L., et al. Effects of shikonin on zebrafish’s embryo and angiogenesis. Chinese Traditional Patent Medicine. 38 (2), 241-245 (2016).
  33. Zhou, Y. . The transformation research on the chemical compositions in the processing of Epimedium. , (2016).
  34. Xiao, Y. P., Zeng, J., Jiao, L. -. N., Xu, X. -. Y. Review for treatment effect and signaling pathway regulation of kidney-tonifying traditional Chinese medicine on osteoporosis. China Journal of Chinese Materia Medica. 43 (1), 21-30 (2018).
  35. Wang, R. H. Study on modern pharmacological effects of traditional Chinese medicine for tonifying kidney yang. Journal of Hubei University of Chinese Medicine. 13 (04), 63-66 (2011).
  36. Luo, L., et al. Advances in the chemical constituents and pharmacological studies of Epimedium. Asia-Pacific Traditional Medicine. 15 (6), 190-194 (2019).
  37. Liu, S., et al. Effects of icariin on ERβ gene expression and serum estradiol level in ovariectomized rats. Hunan Journal of Traditional Chinese Medicine. 32 (1), 150-152 (2016).
  38. Liu, Y., et al. Effects of epimedin A on osteoclasts and osteoporotic male mice. Chinese Journal of Veterinary Science. 41 (07), 1359-1364 (2021).
  39. Liu, Y. L., et al. Effects of icariin and epimedium C on microstructure of bone tissue in glucocorticoid osteoporosis model mice based on Micro-CT technique. Drug Evaluation Research. 43 (09), 1733-1739 (2020).
  40. Zhan, Y. Evaluation of antiosteoporotic activity for micro amount icariin and epimedin B based on the osteoporosis model using zebrafish. Chinese Pharmaceutical Journal. (24), 30-35 (2014).
  41. Zhan, Y., Wei, Y. -. J., Sun, E., Xu, F. -. J., Jia, X. -. B. Two-dimensional zebrafish model combined with hyphenated chromatographic techniques for evaluation anti-osteoporosis activity of epimendin A and its metabolite baohuoside I. Acta Pharmaceutica Sinica. 49 (06), 932-937 (2014).

Tags

Epimedii FoliumMutton OilProcessing TechnologyZebrafish Embryonic DevelopmentResponse Surface MethodBox-Behnken DesignHPLC AnalysisIcariinEAEBECBIComprehensive Score
check_url/pt/65096?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citar este artigo
Fan, J., Wen, X., Li, S., Chu, R., Chen, Y., Su, Z., Li, N. Optimization of the Epimedii Folium Mutton-Oil Processing Technology and Testing Its Effect on Zebrafish Embryonic Development. J. Vis. Exp. (193), e65096, doi:10.3791/65096 (2023).
Baixar citação
Reimpressões e Permissões

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image