这项工作得到了国家自然科学基金（第82130113号）、中国博士后科学基金（编号：2021MD703800）、四川省科技厅青年科学基金（编号：2022NSFSC1449）和成都中医药大学“杏林学者”研究推进计划（No.BSH2021009）。

采用CRITIC结合Box-Behnken响应面法对赞巴炒TBC加工方法进行了优化和标准化。在此过程中，苯甲酰乌头碱、乌头碱和3-脱氧乌头碱用作评价指标。
1. 样品溶液制备
<ol><li>制备对照品储备溶液。在电子分析天平上精确称取9.94 mg苯甲酰乌头碱、8.49 mg乌头碱和6.25 mg3-脱氧乌头碱（材料表），并将它们放入10 mL容量瓶中。然后，加入0.05%盐酸甲醇溶液溶解固体，并将体积补足至10 mL。最后，摇晃混合物，得到质量浓度为0.9940 mg/mL苯甲酰乌头碱、0.8490 mg/mL乌头碱和0.6250 mg/mL 3-脱氧乌头碱的对照物质储备溶液。 注意：盐酸是一种高腐蚀性材料11.使用适当的保护措施，例如手套、实验室外套、护目镜和通风橱。</li>
	<li>准备测试样品溶液。<ol><li>在锥形烧瓶中称取2克Zanba炒TBC粉。<ol><li>称量30克TBC（2厘米）和90克Zanba，然后将它们加入预热的炒菜机中，准备Zanba炒TBC。将炒菜机的时间和温度分别设置为40分钟和140°C。将机器设置为完成处理。</li>
			<li>使用高速粉碎机将 Zanba-炒制的 TBC 分别研磨成粉末样品，这些样品可以通过 50 目（0.355 毫米）的筛子。</li>
		</ol></li>
		<li>根据先前的研究，将3mL氨溶液和50mL异丙醇和乙酸乙酯的混合溶液（比例为1：1 v / v）加入上述锥形烧瓶中12，13。 注意：要制备氨溶液，请将40 mL浓氨溶液加入100 mL容量瓶中，并向测量线中注入纯净水。使用浓氨溶液时采取适当的保护措施，因为它有强烈的气味。</li>
		<li>称取上述样品和锥形瓶并记录重量。超声处理 30分钟 （电压：220 V，频率：40 kHz）。 注意：乌头碱生物碱容易受热分解。因此，超声波提取的温度必须低于25°C。</li>
		<li>超声提取后称取样品和锥形烧瓶。</li>
		<li>通过添加异丙醇和乙酸乙酯的混合物（比例为1：1 v / v）来弥补损失的重量。</li>
		<li>过滤示例溶液。使用旋转蒸发器在40°C下蒸发25mL滤液至干。</li>
		<li>通过加入5mL的0.05%盐酸甲醇溶液溶解残留物，通过0.2μm注射器过滤器过滤溶液，并通过高效液相色谱（HPLC）进行分析。</li>
	</ol></li>
	<li>制备含有0.1988 mg/mL苯甲酰乌头碱、0.0509 mg/mL乌头碱和0.0938 mg/mL 3-脱氧乌头碱的混合参比溶液。 注意：将每种标准品（0.9940mg苯甲酰乌头碱，0.2545mg乌头碱和0.4690mg3-脱氧乌头碱）溶解在0.05%盐酸甲醇中的5mL容量瓶中作为溶解介质。</li>
	<li>通过将6.16g乙酸铵（材料表）溶解在2L超纯水（流动相A）中来制备0.04 M乙酸铵缓冲液。使用氨将pH调节至8.50。 注意：氨是一种有害物质。使用适当的保护措施，例如手套、实验室外套、护目镜和通风橱。</li>
	<li>过滤2L超纯100%乙腈（流动相B）并脱气。 注意：乙腈是一种有害物质13.使用适当的保护措施，例如手套、实验室外套、护目镜和通风橱。</li>
</ol>2. 色谱条件
<ol><li>将 10 μL 预处理的样品溶液注入带有二元泵的 HPLC 系统中。使用采用ODS-3色谱柱（5 μm x 4.6 mm x 250 mm;在30 °C下工作）的HPLC系统，流动相A和B进行MDA和DDA分离。每个样品进样三次以进行技术复制。</li>
	<li>对ODS-3色谱柱的方法进行编程，如 表1 所示。将 流速 设置为 1.0 mL/min ， 检测波长 设置为 235 nm。</li>
	<li>记录每种目标化合物的峰面积。 注意：仪器的详细信息可以在 材料表中找到。</li>
</ol>3. 系统适应性测试
注意：有关执行步骤3.1-3.5的色谱条件，请参阅第2节。
<ol><li>研究浓度与峰面积之间的线性关系。<ol><li>制备各种浓度 - 19.88、39.76、59.64、159.04、198.80 和 497.00 μg/mL - 苯甲酰乌头碱溶液。</li>
		<li>制备各种浓度 - 8.49、16.98、25.47、33.96、50.94 和 169.80 μg/mL - 乌头碱溶液。</li>
		<li>制备各种浓度 - 1.875、12.50、37.50、62.50、93.75 和 125.00 μg/mL - 3-脱氧乌头碱溶液。</li>
		<li>从低质量浓度到高质量浓度进样上述对照品溶液，并记录峰面积。</li>
		<li>从参比溶液浓度（μg/L）与峰面积的关系图中获得三个线性回归方程。 注意：确保苯甲酰乌头碱、乌头碱和 3-脱氧乌头碱的浓度在此标准曲线的线性范围内。</li>
	</ol></li>
	<li>通过将 10 μL 样品溶液连续进样六次重复进行精密测试，并在第 2 节所述的相同 HPLC 条件下运行样品。记录苯甲酰乌头碱、乌头碱和3-脱氧乌头碱的峰面积。</li>
	<li>通过注入10μL制备的样品溶液进行稳定性测试实验，并在0小时，2小时，4小时，8小时，12小时和24小时后测定峰面积。 注意：峰面积由参考的HPLC系统自动记录。这些时间点基于相关文献15，16，17。</li>
	<li>按照步骤1.2中的方法，取同一批Zanba-炒TBC平行制备6种供试样品溶液，进行重现性试验。将每个样品 10 μL 注入 HPLC 系统，并按照第 2 节所述运行样品。 注意：通过比较六个样品之间的浓度差异来评估重现性。</li>
	<li>通过制备六份同批赞巴炒TBC作为供试品溶液来进行回收实验。然后，将各指示组分的~100%对照品加入供试品溶液的六份中，计算回收率。在第 2 节所述的相同条件下将这些样品 （10 μL） 注入 HPLC 系统，并使用公式 （1） 计算回收率： <img alt="Equation 1" src="/files/ftp_upload/65139/65139eq01.jpg" style="margin: auto;">（1） 注：在公式（1）中，A是供试品溶液中待测组分的量，B是添加对照品的量，C是含有对照品和赞巴炒TBC样品的溶液的测量值。</li>
</ol>4. 单因素实验
<ol><li>切片厚度比较<ol><li>准备五组进行测试，每组含 30 克 TBC，其中 TBC 的厚度分别为 0.5、1、2、3 和 4 厘米。称量赞巴的量是TBC（90克）的三倍。 注意：待定是有毒的。使用适当的保护措施，例如手套、实验室外套、护目镜和通风橱，并在切割过程中小心。通过前期实验，发现TBC和赞巴完全接触所需的赞巴量是其三倍。因此，在正式的实验设计中，研究在检查切片厚度时选择了三倍的Zanba量。</li>
		<li>将自动炒菜机的温度和时间分别设置为 140°C 和 40分钟。</li>
		<li>在自动炒菜机加热到设定温度后，将~30克TBC和90克Zanba加入机器中。</li>
		<li>按照步骤 1.2 准备示例解决方案。根据标准曲线计算不同加工产品中MDA和DDA的含量（表2）。通过第 6 节中的 CRITIC 方法根据结果计算综合分数。</li>
		<li>通过这种方式，比较赞巴的量，以及加工温度和时间，以优化条件。</li>
	</ol></li>
	<li>赞巴的量比较<ol><li>进行五组测试，每组含 30 克 TBC（2 厘米），其中 Zanba 的含量分别为 TBC 的 1 倍、2 倍、3 倍、4 倍和 5 倍。</li>
		<li>打开炒菜机进行处理。将炒菜机的时间和温度设置为 40分钟 和 140°C。</li>
		<li>按照步骤 1.2 准备示例解决方案。根据标准曲线计算不同加工产品中MDA和DDA的含量（表2）。通过第 6 节中的 CRITIC 方法 根据 结果计算综合分数。</li>
	</ol></li>
	<li>加工温度比较<ol><li>进行五组测试，每组用30克TBC（2厘米）和90克赞巴。</li>
		<li>打开炒菜机进行处理。将处理温度设置为 100 °C、120 °C、140 °C、160 °C 和 180 °C。 将处理时间设置为 40 分钟。 注意：通过前期实验发现，加工温度低于100°C时，赞巴黄变的速度很低，如果温度过高（180°C以上），赞巴容易燃烧变黑。因此，100 °C和180 °C分别设置为加工过程中的最小和最高温度值。</li>
		<li>按照步骤 1.2 准备示例解决方案。记录MDA和DDA的峰面积。根据标准曲线计算不同加工产品中MDA和DDA的含量（表2）。通过第 6 节中的 CRITIC 方法 根据 结果计算综合分数。 注意：该实验涉及160°C和180°C的高温。 根据实验室的安全规范，在实验过程中注意安全。</li>
	</ol></li>
	<li>处理时间比较<ol><li>进行五组测试，每组用30克TBC（2厘米）和90克赞巴。</li>
		<li>打开炒菜机进行处理。将处理时间设置为 20、40、60、80 和 100 分钟。将温度设置为 140 °C。</li>
		<li>按照步骤 1.2 中的说明准备示例解决方案。记录MDA和DDA的峰面积。根据标准曲线计算不同加工产品中MDA和DDA的质量（表2）。通过第 6 节中的 CRITIC 方法 根据 结果计算综合分数。</li>
	</ol></li>
</ol>5. 基于响应面法（RSM）的炒TBC加工工艺优化
<ol><li>博克斯-本肯响应面设计<ol><li>通过使用单因素测试（步骤4.1-4.4）的初步实验，确定切片厚度的范围（A，1-3厘米），赞巴的量（B，2-4x），处理温度（C，100-140°C）和处理时间（D，40-80分钟）。 注意：四个变量的编码值及其电平如 表3所示。每个变量的三个级别分别编码为 -1、0 和 1。</li>
	</ol></li>
	<li>使用软件生成矩阵并分析响应面模型。 注：软件使用情况的屏幕截图显示在 补充文件 1 中。<ol><li>使用由 24 个实验组成的三水平四因子 Box-Behnken 设计（如本研究中所做的那样），并测量五个重复（运行顺序 1、9、14、16 和 25）来计算平方的纯误差和（表 4）。将综合分数 （Y） 设置为响应（步骤 1-4， 补充文件 1）。<ol><li>在 主页上，单击“ 新建设计 ”（步骤 1， 补充文件 1），然后在“ 设计 ”页面的左侧面板中，单击 “响应面 |Box-Behnken 并设置表中四个因子的参数（步骤 2， 补充文件 1）。</li>
			<li>单击下一步（步骤 2，补充文件 1），设置响应名称，然后单击完成（步骤 3，补充文件 1）。 </li>
			<li>通过上述操作生成响应面设计（步骤 4， 补充文件 1）。</li>
		</ol></li>
	</ol></li>
	<li>根据为响应曲面设计的 29 个方案完成试验。</li>
	<li>按照步骤 1.2 准备示例解决方案。</li>
	<li>记录MDA和DDA的峰面积。 注意：峰面积由参考的HPLC系统自动记录。</li>
	<li>计算不同加工产品中MDA和DDA的质量。</li>
	<li>通过步骤 6 中的 CRITIC 方法 根据 结果计算综合分数。 注意：具体方法如步骤 6 所示。</li>
	<li>将获得的29次试验的综合分数输入计算机，并使用参考软件进行分析（步骤5， 补充文件1）。</li>
	<li>通过软件对绘制在 3D 模型图中的多项式方程和响应面分析进行统计验证（步骤 6-8， 补充文件 1）。<ol><li>在左侧导航窗格中的“分析 （+）”下，单击 Y，然后单击“配置”窗口中的“开始分析”（步骤 6，补充文件 1）。</li>
		<li>单击顶部菜单中的方差分析，观察显示 方差分析 的结果表（步骤 7， 补充文件 1）。</li>
		<li>在顶部菜单中，单击 模型图 ，然后单击 3D 表面 以获取反映处理参数对合成分数影响的响应曲面图（步骤 8， 补充文件 1）。</li>
	</ol></li>
	<li>在预测的最佳条件下（步骤9， 补充文件1）一式三份对响应面模型进行验证，以验证加工技术的稳定性。在左侧 导航 窗格中的 优化下，单击 数字 ，然后在顶部菜单中单击 解决方案。观察预测的最佳条件。</li>
</ol>6. 模型评估
注意：此步骤在每个单因素实验或响应面实验完成后执行。每个实验（例如，切片厚度的比较）完成后，根据步骤1.2和第2节测量不同样品中MDA和DDA的含量，以获得五个数据集。数据见 补充表S1。
<ol><li>指数的无量纲处理 注意：此步骤将测量值（Xij）转换为无量纲相对值，以便每个指数的值处于相同的数量水平。此操作可以便于对不同单位或数量级的指标进行综合分析和比较18.为了便于说明，切片厚度值已用于如下所示的计算（补充表S1）。<ol><li>规范MDA的内容（获得yMDA;MDA是指苯甲酰乌头碱），通过使用公式（2）中的公式。 注意：索引“i”代表四个因素之一，切片厚度是研究的第一个因素。因此，i 的值等于 1。指数“j”代表每个水平的因素;因此，当切片厚度为第一级（0.5厘米）时，J等于1;当切片厚度为第五级（4厘米）时，j等于5。厚度为0.5、1、2、3和4 cm的TBC中MDA（Xij）含量分别为0.9693、1.0876、1.3940、1.4185和1.3614 mg/g。因此，x j，最大值 为 1.4185，xj，最小 值为 0.9693。 <img alt="Equation 2" src="/files/ftp_upload/65139/65139eq02.jpg" style="margin: auto;">（二） 因此 <img alt="Equation 3" src="/files/ftp_upload/65139/65139eq03.jpg" style="margin: auto;"> 这里，Xij 是实验在第i个因子和第j个水平上的MDA的测量内容;xj，min 是本组实验中MDA的最小含量;和 xj，max 是这组实验中 MDA 的最大含量。因此， i = 1， 2， ...， m 和 j = 1， 2， ...， n。 注意： 因此，MDA 的标准化值是 0.0000、0.2634、0.9455、1.0000 和 0.8729，使用公式 （2）。</li>
		<li>规范DDA的总内容（获得yDDA;DDAs是指乌头碱和3-脱氧乌头碱），使用公式（3）中的公式。 注意： i 是四个因素之一， j 是 每个水平的因素;Xij 是实验的DDA在第i个因子和第j个水平上的测量内容;xj，min 是本组数据实验中DDAs的最小含量;而xj，max是 本组数据实验中DDA的最大含量。这样， i = 1， 2， ...， m 和 j = 1， 2， ...， n。厚度为0.5、1、2、3和4 cm的TBC中DDAs（Xij）含量分别为0.3492、0.2692、0.2962、0.5354和0.5124 mg/g。因此，x j，最大值 为 0.5354，xj，最小 值为 0.2692。 <img alt="Equation 4" src="/files/ftp_upload/65139/65139eq04.jpg" style="margin: auto;">（三） <img alt="Equation 5" src="/files/ftp_upload/65139/65139eq05.jpg" style="margin: auto;"> 注意： 使用等式 （3） 的标准化值为 0.6995、1.0000、0.8986、0.0000 和 0.0864。</li>
	</ol></li>
	<li>根据方程计算相应的对比度强度（S i）、冲突（δ i）、信息（C i）和指数权重（W i）。（4）至（7）分别为19、20。 注意：i = 1， 2， ...， m.y ij 是第 i 个因子和第 j 个因子实验的 MDA 或 DDA 含量的标准化数据。<ol><li>要估计对比度强度，请首先计算平均MDA值。 <img alt="Equation 6" src="/files/ftp_upload/65139/65139eq06.jpg" style="margin: auto;"> MDA的平均值在哪里 <img alt="Equation 7" src="/files/ftp_upload/65139/65139eq07.jpg" style="margin: auto;">。 <img alt="Equation 8" src="/files/ftp_upload/65139/65139eq08.jpg" style="margin: auto;">（四） <img alt="Equation 21" src="/files/ftp_upload/65139/65139eq21v4.png" style="margin: auto;"></li>
		<li>要计算冲突值，请首先使用 Excel21 中的 CORREL 函数估计ij γ相关系数。 <img alt="Equation 9" src="/files/ftp_upload/65139/65139eq09.jpg" style="margin: auto;">（五） <img alt="Equation 10" src="/files/ftp_upload/65139/65139eq10.jpg" style="margin: auto;"></li>
		<li>按如下方式计算信息值。 <img alt="Equation 11" src="/files/ftp_upload/65139/65139eq11.jpg" style="margin: auto;">（六） <img alt="Equation 12" src="/files/ftp_upload/65139/65139eq12.jpg" style="margin: auto;"> 注意：同样，C1，DDAS = 0.7210</li>
		<li>按如下方式计算指数权重。 <img alt="Equation 13" src="/files/ftp_upload/65139/65139eq13.jpg" style="margin: auto;">（七） <img alt="Equation 14" src="/files/ftp_upload/65139/65139eq14.jpg" style="margin: auto;"> 注意：因此，MDA和DDAs在比较切片厚度时的重量系数分别确定为0.4945和0.5055。</li>
	</ol></li>
	<li>计算切片厚度的综合得分。 <img alt="Equation 15" src="/files/ftp_upload/65139/65139eq15.jpg" style="margin: auto;"> <img alt="Equation 16" src="/files/ftp_upload/65139/65139eq16.jpg" style="margin: auto;"> <img alt="Equation 17" src="/files/ftp_upload/65139/65139eq17.jpg" style="margin: auto;"> <img alt="Equation 18" src="/files/ftp_upload/65139/65139eq18.jpg" style="margin: auto;"> <img alt="Equation 19" src="/files/ftp_upload/65139/65139eq19.jpg" style="margin: auto;"> 注意：Y13 是最大值。因此，切片厚度的最佳参数是第三级 - 2 厘米。</li>
</ol>

一种 Zanba-Stir-Fried Tiebangchui 工艺优化方法

<ol>
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作者没有利益冲突需要披露。

TBC是一种重要的藏药，具有祛寒止痛的作用。数千年来，它在中国主要用于治疗创伤性损伤和风湿性关节痛24，25，26。二萜类生物碱是TBC 27，28，29的活性和毒性成分。TBC的乌头生物碱的主要毒性作用是神经毒性、心脏毒性和胃肠道毒性30<s...

乌头摆布希和黄头的干根，最著名的藏药之一，在中国1，2中被称为铁棒翠（TBC）。TBC的干燥根有助于驱寒和风，减轻疼痛和镇静休克。记录在《中华人民共和国卫生部药品标准（藏药）》第一卷中，其中指出TBC的干根通常用于治疗类风湿性关节炎，跌打损伤和其他感冒疾病3。但TBC的临床治疗剂量与其毒性剂量相似，由于使用不当，中毒或死亡事件屡见不鲜有报道4。因此，降低TBC的毒性，保持疗效成为多年来的研究热点。
在藏药中，加工是减轻TBC毒性的最有效方法之一。根据《青海省藏药加工规范（2010）》，原药材（待定）放入铁锅中，与赞巴一起炒至赞巴变黄，取出赞巴，空气晒干5，6。然而，没有具体的工艺参数记录，这使得控制加工技术和Zanba炒TBC的质量变得困难。CRITIC法是一种客观的权重方法，可以避免模糊化和主观性，增强称重的客观性7.Box-Behnken响应面法可以通过多项式拟合8直接反映各因子之间的相互作用。Box-Behnken响应面和CRITIC方法的组合通常用于优化处理技术，以获得优化的处理协议9，10。本文以单酯-二萜类生物碱（MDA）（苯甲酰乌头碱）和两种二酯-二萜类生物碱（DDAs）（乌头碱、3-脱氧乌头碱）为评价指标。采用CRITIC结合Box-Behnken响应面法优化赞巴炒TBC加工工艺，建立临床安全使用标准加工方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-Deoxyaconitine</td>
			<td>Chengdu Desite Biotechnology Co., Ltd.</td>
			<td>DST221109-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Aconitine</td>
			<td>Chengdu Desite Biotechnology Co., Ltd.</td>
			<td>DSTDW000602</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>Tianjin Kermel Chemical Reagent Co., Ltd</td>
			<td>Chromatographic grade</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzoylaconitine</td>
			<td>Chengdu Desite Biotechnology Co., Ltd.</td>
			<td>DSTDB005502</td>
			<td></td>
		</tr>
		<tr>
			<td>Design-Expert software</td>
			<td>Stat-Ease, Inc., Minneapolis, MN, USA</td>
			<td>version 13.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic analytical balance</td>
			<td>Shanghai Liangping Instruments Co., Ltd.</td>
			<td>FA1004</td>
			<td></td>
		</tr>
		<tr>
			<td>High performance liquid chromatography</td>
			<td>SHIMADZU Co., Ltd.</td>
			<td>LC-20A</td>
			<td></td>
		</tr>
		<tr>
			<td>High-speed smashing machine</td>
			<td>Beijing Zhongxing Weiye Instrument Co., Ltd.</td>
			<td>FW-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore filter</td>
			<td>Tianjin Jinteng Experimental Equipment Co., Ltd</td>
			<td>&#966;13 0.22 Nylon66</td>
			<td></td>
		</tr>
		<tr>
			<td>stir-Fry machine</td>
			<td>Changzhou Maisi Machinery Co., Ltd</td>
			<td>Type 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tiebangchui</td>
			<td>Gannan Baicao Biotechnology Development Co., Ltd</td>
			<td>20211012</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra pure water systemic</td>
			<td>RephiLe Bioscience, Ltd.</td>
			<td>Genie G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleansing machine</td>
			<td>Ningbo Xinyi Ultrasonic Equipment Co., Ltd</td>
			<td>SB2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Zanba</td>
			<td>27 Chuanzang Road, Ganzi County</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimization of processing technology for tiebangchui with zanba based on critic combined with box-behnken response surface method

乌头摆的干根，中文称为铁棒翠（TBC），是最著名的藏药之一。它是中国西北地区广泛使用的草药。然而，由于TBC的强烈毒性以及其治疗剂量和毒性剂量相似，发生了许多中毒病例。因此，寻找一种安全有效的方法来降低其毒性是一项紧迫的任务。翻阅藏药典籍，《青海省藏药加工规范（2010）》中记载了TBC炖炒的加工方法。但是，具体的加工参数尚不清楚。因此，本研究旨在优化和规范赞巴炒TBC的加工工艺。
首先，对TBC切片厚度、Zanba用量、加工温度和时间4个因素进行单因素试验。以赞巴炒TBC中单酯和二酯生物碱含量为指标，采用CRITIC结合Box-Behnken响应面法优化赞巴炒TBC的加工工艺。赞巴炒TBC的优化加工条件为TBC切片厚度为2 cm，赞巴是TBC的3倍，加工温度为125 °C，翻炒60 min。本研究确定了赞巴炒TBC使用的优化和标准加工条件，从而为赞巴炒TBC的安全临床使用和工业化生产提供了实验依据。

The dried root of Aconitum pendulum Busch and A. flavum Hand.-Mazz., one of the most famous Tibetan medicines, is called Tiebangchui (TBC) in Chinese1,2. The dried roots of TBC are helpful in dispelling cold and wind, reducing pain, and calming shock. It was recorded in the first volume of "Drug Standards (Tibetan Medicine) of the Ministry of Health of the People's Republic of China," which states that the dried roots of TBC are commonly used to treat rheumatoid arthritis, bruises, and other cold diseases3. However, the clinical therapeutic dose of TBC is similar to its toxic dose, and incidents of poisoning or death have been frequently reported due to improper use4. Therefore, reducing the toxicity and preserving the efficacy of TBC has become a research hot spot over the years.
In Tibetan medicine, processing is one of the most effective methods to attenuate the toxicity of TBC. According to "Processing specification of Tibetan medicine of Qinghai Province (2010)", the original herbs (TBC) should be placed in an iron pot and stir-fried with Zanba until the Zanba turns yellow, after which Zanba is removed and the herbs are dried in air5,6. However, no specific process parameters have been documented, which makes controlling the processing technology and the quality of Zanba-stir-fried TBC difficult. The CRITIC method is an objective weight method that can avoid fuzzification and subjectivity, and enhance the objectivity of weighing7. The Box-Behnken response surface method can directly reflect the interaction between each factor through polynomial fitting8. The combination of the Box-Behnken response surface and CRITIC method is commonly used to optimize processing technology to acquire the optimized processing protocol9,10. In this paper, a monoester-diterpenoid alkaloid (MDA) (benzoylaconitine) and two diester-diterpenoid alkaloids (DDAs) (aconitine, 3-deoxyaconitine) were used as evaluation indexes. CRITIC combined with the Box-Behnken response surface method was applied to optimize the processing technology of Zanba-stir-fried TBC and establish a standard processing method for clinical safe use.

作者聚焦：基于 CRITIC 结合 Box-Behnken 响应面法的 Tiebangchui 与 Zanba 加工工艺优化  

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 scope of this protocol lies in the proceeding of the Tibetan medicines. This staff aims to the optimize and standardize the processing technology of Zanba-stir-fried Tiebangchui. The combination of the Box-Behnken response surface and the CRITIC method is commonly used to optimize proceeding technology to acquire the optimized proceeding protocol.The optimized processing conditions of two centimeter Tiebangchui slice thickness three times more Zanba than Tiebangchui, a processing temperature of 125 degrees C and the 60 metres of stir-frying. By quantifying the specific processing parameters, this method will benefit the large-scale production and the quantity control of Zanba-stir-fried Tiebangchui. The standard Zanba-stir-fried processing technology of Tiebangchui can provide useful information and the guidance for the processing of other poisonous ethnical medicines.The optimized process is stable and feasible, laying a certain foundation for further investigation of Tiebangchui products and related Chinese medicine preparations. Our laboratory will pay more attention to cytochemical investigation to obtain the targeted quantity control compliance. In addition, the pharmacological comparison overall in Zanba-stir-fried Tiebangchui is unclear.Detoxification and evaluation of the efficacy, reservation effects in animal models will be the next objectives.

在这项研究中，使用的洗脱梯度对Zanba-炒-炒TBC中的三种指数组分具有良好的分辨率（图1），这是在重复调试后确定的。三霸炒TBC中的3种指标组分在特定浓度范围内具有良好的线性关系（表2）。炒TBC的精密度（表5）、稳定性（表6）、重复性（表7）和样品回收率（表8）均在中国药典（2020年第4卷）22规定?...

Watch this Scientific Journal Video about Optimization of Processing Technology for Tiebangchui with Zanba Based on CRITIC Combined with Box-Behnken Response Surface Method at JoVE.com

本协议描述了一种使用CRITIC结合Box-Behnken反应面法的Zanba炒铁棒净的高效和标准的解毒处理方法。

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 ...

该议定书的范围在于藏药的进行。该团队旨在优化和规范赞巴炒铁板岀的加工工艺。Box-Behnken响应面和CRITIC方法的组合通常用于优化程序技术，以获得优化的程序方案。优化加工条件，两厘米铁板岔片厚度是铁板岔的三倍，加工温度125°C，60米的炒制。通过量化具体的加工参数，该方法将有利于三霸炒铁房滓的大规模生产和数量控制。铁棒滔标准的炒加工工艺可以为其他有毒民族药物的加工提供有用的信息和指导。优化后的工艺稳定可行，为进一步研究铁棒翠产品及相关中药制剂奠定了一定基础。我们的实验室将更加重视细胞化学研究，以获得目标数量控制合规性。此外，赞巴炒铁房翠的整体药理比较尚不清楚。排毒和评价动物模型中的疗效、保留效果将是下一个目标。

optimization-processing-technology-for-tiebangchui-with-zanba-based

Research

JoVE Journal

Medicine

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

716 次观看.  Chengdu University of Traditional Chinese Medicine. 该议定书的范围在于藏药的进行。该团队旨在优化和规范赞巴炒铁板岀的加工工艺。Box-Behnken响应面和CRITIC方法的组合通常用于优化程序技术，以获得优化的程序方案。优化加工条件，两厘米铁板岔片厚度是铁板岔的三倍，加工温度125°C，60米的炒制。通过量化具体的加工参数，该方法将有利于三霸炒铁房滓的大规模生产和数量控制。铁棒滔标准的炒加工工艺可以为其他...

视频: 作者聚焦：基于 CRITIC 结合 Box-Behnken 响应面法的 Tiebangchui 与 Zanba 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 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.

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.

科研

医学

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.

制备三叶炒铁棒撀（TBC）样品进行加工技术优化

Response Surface Methodology (RSM) for Processing Technology Optimization of Zanba-Stir-Fried Tiebangchui (TBC)

To perform the Box-Behnken in response surface design for optimization of Zanba-Stir-Fried Tiebangchui or TBC processing technology. Use the range of sliced thickness the amount of Zanba, the processing temperature and the processing time determined by preliminary experiments using single factor tests. On the homepage.Click on new design, and in the left panel of the design page, click on response surface. Then click Box-Behnken and set the parameters for the four factors in the table. Click on next, set the response names and click on finish.To generate the response surface design. Complete the experiment based on the 29 scenarios designed for the response surface, using high performance liquid chromatography or HPLC. Record the peak areas and determine the quality of the MDA and DDAs in the Zanba Stir-fried TBC test samples.Then input the comprehensive scores obtained via the critic method for the 29 trials into the computer and analyze it using the referenced software, to perform the statistical validation of the polynomial equations in the left navigation pane. Under analysis plus, click on R1 and then click on start analysis. In the configure window, click on ANOVA in the top menu and observe the results table displaying variance analysis.Next in the top menu, click on model graphs and then 3D surface to obtain the response surface plots reflecting the effects of processing parameters on the synthetic scores. Go to the left navigation pane and under optimization click on numerical to validate the response surface model in triplicate under the predicted optimum conditions. Then in the top menu, click on solutions observe the predicted optimal conditions to verify the stability of the processing technology.The response surfaces and contour plots demonstrated the changes in synthetic scores as a function of four variables, on the basis of the experimental results. The adjusted optimal processing conditions were a slice thickness of two centimeters, three times more Zanba than TBC, a processing temperature of 125 degrees Celsius and a processing time of 60 minutes. The reliability of the model was proven through three tests that were conducted according to the obtained processing parameters.