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

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。

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。
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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>MUST-20080403</td>
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			<td>Chengdu Manst Biotechnology Co., Ltd.</td>
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			<td>6.02</td>
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			<td>Tianjin Jinteng Experimental Equipment Co., Ltd.</td>
			<td>0.22&#956;m</td>
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			<td>20211106</td>
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			<td>China Zebrafish Resource Center (CZRC)</td>
			<td>The AB strain</td>
			<td></td>
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	</tbody>
</table>

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

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。

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...

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

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。

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

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 ...

在这项研究中，我们采用了一种广泛使用的加工方法，并获得了一种稳定可靠的持久技术。结果表明，加工后的淫羊藿毒性降低。响应面法需要更少的实验和更短的周期，既避免了传统数理统计中存在的问题，又有助于用户选择最优条件。该方法还可广泛应用于化工、生物工程、食品工业、中药制剂、中药提取等。制备溶液并进行色谱分析后，选择EF处理中的关键参数，如羊油的量，羊油的温度和煎炸的温度作为影响因素。使用淫羊藿苷、EA、EB、EC和BI含量的综合得分作为评价指标。使用响应面分析软件设计Box-Behnken响应面实验，探索二次响应面，并构建二阶多项式模型。选择新的 Box-Behnken 设计，并将数值因子选项设置为 3。设置因子 A、B 和 C。单击继续，然后将响应选项设置为 1，然后单击继续以完成设计。根据具体参数处理EF，例如，对于订单号一，精炼羊油按体积的15%体积，然后将其加热到50摄氏度使其融化。将粗EF加入融化的羊肉中。用温和的火翻炒至均匀光泽，然后取出并冷却EF。准备加工产品的测试溶液，并按照手稿中的说明使用HPLC进行分析。记录每种化学成分的保留时间和峰面积，并根据外部标准曲线计算每种测试溶液中淫羊藿苷、EA、EB、EC和BI的含量。采用综合分数计算公式计算17个实验组的综合得分。将17组实验的综合评分结果导入数据分析软件，对实验数据进行分析。在评估项下，选择二次处理顺序选项和多项式模型类型选项。与其他供试因子相比，羊油用量和油炸温度的响应面更陡峭，等高线趋于椭圆形，表明这两个因子之间的交互作用比其他因子之间的交互作用更显著。混合对照品、淫羊藿粗品和淫羊藿叶处理品的HPLC色谱图表明，原始EF中的BI含量较低，但加工后增加。粗品组在150μg/毫升药物浓度下表现出明显的脊柱畸形、身体弯曲、心包水肿和肝脏变形，而加工组幼鱼的这些变化很少见。生药组所有斑马鱼在给药200微克/毫升后24小时死亡，而加工组死亡率仅为6.67%每毫升200微克EF导致生药组所有斑马鱼在72小时后死亡，而在加工组，每毫升EF500微克导致所有斑马鱼死亡。两个实验组在受精后72 h的中位致死浓度显示，粗组LC 50为151.3 μg/毫升，加工组为219.8 μg/毫升。实验中最重要的是选择响应面法的因子，并确定这些因子的参数。

Research

JoVE Journal

Medicine

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

比浊法对人洗涤血小板：所述Pannexin1抑制剂的影响亮蓝FCF对胶原诱导的聚集

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

科研

医学

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

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

测定化学物质对线虫线虫生长和繁殖的影响

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

痰液诱导和实验室处理方法

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

一种定量感官检测方法在乳腺癌手术患者疼痛处理中的研究

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

青少年及有脑震荡青少年多学科技术强化护理途径的开发与实施

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

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

测定细菌和化学物质对开牛结不二病的肠道渗透性的影响

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

研究辅助供氧对囊性纤维化气道微生物组影响的模型的设计与开发

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

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

基于CRITIC结合Box-Behnken响应面法的铁邦岀与赞坝加工工艺优化

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

基于Box-Behnken设计结合熵法的高原青稞酒铁板廚加工优化

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