基于Box-Behnken设计结合熵法的高原青稞酒铁板廚加工优化
Summary
本协议描述了一种基于Box-Behnken设计响应面结合熵法优化高原大麦酒铁棒翠加工工艺的有效方法。
Abstract
有毒民族药物的加工对其安全临床应用具有重要意义。因此，应解决传统加工的局限性，并应使用现代研究方法对民族药物的加工方法进行标准化。本研究对常用藏药铁板翠（TBC）的加工工艺进行了优化，即 乌头摆布希的干根，用高原大麦酒加工而成。以二酯-二萜类生物碱（DDA）（乌头碱、3-脱氧乌头碱、3-乙酰乌头碱）和单酯-二萜类生物碱（MDA）（苯甲酰乌头碱）含量为评价指标，采用熵法确定各评价指标的权重系数。
采用单因素试验和Box-Behnken设计，研究了高原青稞酒与TBC配比、TBC切片厚度和加工时间的影响。根据熵法确定的各指标的客观权重进行综合评分。TBC与高原青稞酒的最佳加工条件为：高原青稞酒用量是TBC的5倍，浸泡时间为24 h，TBC厚度为1.5 cm。结果表明，验证试验与预测值的相对标准偏差小于2.55%，高原青稞酒TBC加工优化工艺简单、可行、稳定，可为工业化生产提供参考。
Introduction
铁板翠（TBC）是乌头摆布希的干根，是一种著名的藏药，最初记录在经典藏医书籍“四药密宗”1,2中。根据《中华人民共和国卫生部药品标准（藏医）》，TBC具有祛寒、止痛、祛风、镇静休克等功效，常用于治疗临床类风湿性关节炎3、4、5。
TBC主要含有生物碱，包括剧毒的二酯-二萜类生物碱（DDAs）和中毒性单酯-二萜类生物碱（MDAs）6,7,8。这些化学成分是具有药用作用的活性成分，但有毒。最著名的活性和有毒成分之一乌头碱在超过1mg9时会引起中毒。因此，TBC使用不当或过量可能导致中毒甚至死亡，TBC的毒性减弱和疗效储备对其安全临床应用至关重要10,11。
处理是排毒TBC的有效方法。据古代藏医书籍记载，用高原大麦酒加工是减轻毒性、保持TBC功效的有效方法。TBC浸泡在高原大麦酒中，储存一晚，干燥，并添加到药物中12。然而，具体的加工工艺和潜在影响因素很少报道，传统的加工工艺往往依赖经验，缺乏标准化的方法。因此，需要现代科学技术方法来优化和标准化加工过程。
Box-Behnken设计方法用于通过二次多项式拟合研究不同因素之间的相互作用及其对综合评分的影响。这种设计允许直观地观察最佳条件，并已广泛应用于药学领域13。例如，基于熵法的Box-Behnken设计方法成功优化了姜黄14醋炒加工工艺。本研究采用Box-Behnken响应面实验设计结合熵法优化了高原大麦酒TBC加工工艺。优化的加工技术有望确保质量控制和安全的临床使用。
Protocol
本研究采用Box-Behnken设计结合熵法优化了高原青稞酒TBC加工工艺。以DDA和MDA含量为评价指标，采用熵法确定各评价指标的权重系数。 1. 实验准备 准备高原大麦酒15.取500.00克黑高原大麦米，加入五倍量的水。煮米饭直到剩余的水被吸收（~2小时）。倒出，待温度降至37°C，加入酒曲4克（见 材料表），拌匀，封罐，用棉絮?…
Representative Results
本研究对TBC的精密度、稳定性、重复性和样品回收率表明该方法是可行的。TBC中的4种指标组分在特定浓度范围内具有良好的线性关系。典型色谱图如图 1所示。精密度测试结果（表8）显示，苯甲酰乌头碱、乌头碱和3-脱氧乌头碱峰面积的相对标准偏差（RSD）分别为2.56%、1.49%和2.03%，3-乙酰乌头碱的峰面积相对标准偏差（RSD）分别为0.21%，表明仪器精密度良好。进?…
Discussion
作为一种具有毒性作用的常用藏药，加工的毒性减弱作用对TBC的临床应用极为重要25。本研究优化了高原青稞酒TBC加工工艺。通过回顾TBC的主要活性成分并关联TBC的药理作用，我们发现TBC生物碱具有抗炎和镇痛作用，可用于治疗类风湿性关节炎。在这项研究中，使用中毒性MDA作为阳性指标。DDA的总含量被用作负面指标。采用熵法计算指标权重，优化处理工艺26。…
Declarações
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| Aconitine | Chengdu Push Biotechnology Co.,Ltd | PS000905 | |
| 3-Acetylaconitine | Chengdu Push Biotechnology Co.,Ltd | PS010552 | |
| 3-Deoxyaconitine | Chengdu Push Biotechnology Co.,Ltd | PS011258 | |
| Benzoylaconine | Chengdu Push Biotechnology Co.,Ltd | PS010300 | |
| Circulating water vacuum pump | Gongyi City Yuhua Instrument Co., Ltd | SHZ-DIII | |
| Design-Expert | State-East Corporation | 8.0.6 | |
| Electric constant temperature drying oven | Shanghai Yuejin Medical Equipment Co., Ltd | 101-3-BS | |
| Electronic analytical balance | Shanghai Liangping Instruments Co., Ltd. | FA1004 | |
| High performance liquid chromatography | Shimadzu Enterprise Management (China) Co., Ltd | shimadzu 2030 | |
| Highland barley rice | Kangding City, Ganzi Tibetan Autonomous Prefecture, Sichuan Province | 20221015 | |
| Millipore filter | Tianjin Jinteng Experimental Equipment Co., Ltd | φ13 0.22 Nylon66 | |
| Rotary evaporator | Shanghai Yarong Biochemical Instrument Factory | RE-2000A | |
| Starter of liquor-making | Angel Yeast CO., Ltd | BJ22-104 | |
| Ultra pure water systemic | Merck Millipore Ltd. | Milli-Q | |
| Ultrasonic cleansing machine | Ningbo Xinyi Ultrasonic Equipment Co., Ltd | SB-8200 DTS |
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Citar este artigo
Yu, L., Li, S., Tan, X., Wang, C., Lai, X., Liu, Y., Zhang, Y. Optimization of Processing of Tiebangchui with Highland Barley Wine Based on the Box-Behnken Design Combined with the Entropy Method. J. Vis. Exp. (195), e65154, doi:10.3791/65154 (2023).