草药的制备：洱西汤和含茜素的血清<em>体内</em>和<em>体外</em>实验
Summary
在这里，我们以四个步骤 – 浸泡，煎煮，过滤和浓缩四个步骤介绍了一种呃ian汤（EXD）的制备方法，并且证明了给大鼠制备的含有EXD的血清。这些方法适用于中药等中草药的体内和体外研究。
Abstract
传统草药是临床环境中的替代药物，近年来受到越来越多的关注。在运送到身体之前，通常需要额外的提取方法来从原料中释放活性成分。水煎剂是一种经典的提取方法，在临床环境中仍然广泛应用。在这里，我们提出了一个详细的呃ian汤（EXD）方案，以便将草药煎药应用于实验研究。描述了动物适当剂量的计算，以及EXD的四个主要步骤：浸泡，水煎剂，过滤和浓缩。此外，将含有血清的EXD作为体外验证手段引入大鼠。在这里，大鼠经口给予EXD三天。然后收集血液样品，灭活，离心和过滤。用培养基稀释的血清可用于治疗v。中的细胞或组织itro。例如，EXD被应用于体内和体外研究，并且表明EXD增强成骨。该方案可作为草药的制备和应用参考。
Introduction
对传统草药研究和应用的兴趣目前正在增长。与现代药物相反，化学成分明确，草药配方有一些未知的成分，需要提取过程才能使其活性化合物交付。虽然许多研究尝试选择一种具有众所周知结构的小化合物作为全草药或草药配方的代表，但药理作用和机制均不能等同于1,2 。虽然指纹图谱能够分析复方草药配方的成分，但一些成分仍未得到清晰分析，结合所有研究提取物3时会带来挑战。许多成分/提取物的相互作用介导中草药的治疗效果。为了保留这个优势，传统的提取物形式 – 煎煮离子 – 仍然广泛应用于诊所。由于提取方法对治疗效果有很大影响，因此传统水煎剂的标准方案是必要的，特别是对于体内研究4,5 。
另一方面，当探索药理机制时，体外或体外研究中草药煎剂的施用也是一个挑战。药物血清的概念最初由Tashino于1988年提出6 。从那时起，越来越多的研究人员将其应用于草药7,8,9 。虽然含药血清的方法有一定的局限性，如血清某些成分的影响，但仍被认为是与生理条件密切相关的方法。
<p class="“jove_content”">二十世纪五十年代初开发的二味汤是为了缓解更年期症状10,11,12,13。它也被应用于治疗再生障碍性贫血14 ，更年期骨质疏松症15,16,17 ，早产卵巢功能衰竭18 ，乳腺癌19 ，卵巢癌20和延迟青春期21 。在这里，我们提出了制定呃ian汤（EXD）及其含药血清的详细方案。此外，我们描述了含有EXD和EXD的血清对鼠绝经期骨质疏松模型的应用。一个EXD由9g Curculigo orchioides Gaertn ， Herbaa Epimedii组成 </em + ， 甘草和当归，每个成年患者每天6g 皮质黄柏和根瘤菌 。基于以下等式22，小鼠的等效剂量为0.1418EXD / kg /天：dB = dA * RB / RA *（WA / WB） 1/3 。 dA和dB分别表示人和小鼠每体重的剂量（mg / kg）。剂量（mg / kg）由EXD / kg的数量代替。 RA和RB分别代表（体表面积（m 2 ）/体重（kg）） 2/3 （ 参见表1 ）成比例的人体因子和小鼠体内因子。 WA和WB分别表示人和小鼠的体重（kg）。
Protocol
该协议遵循上海中医药大学动物护理方针，经动物实验上海动物伦理委员会批准。 议定书I：准备EXD 计算 将EXD给予10名6个月大的雌性印记控制区（ICR）小鼠（0.02kg /小鼠）的治疗组，每周5天，持续12周。 注意：需要总共1.702个EXD。因为EXD是以离散的增量来衡量的，在实际中，管理2个EXD。 计算每只小鼠施用的EXD的体积（ 即 V =（0.141…
Representative Results
EXD对OVX小鼠骨密度的影响腰椎椎体部分的苏木精和伊红（H＆E）染色显示体外EXD处理后增加的骨小梁（ 图1A ，右图）与OVX组相比（ 图1A ，左图）。 图1B显示OVX小鼠（ 图1B ，左图）和EXD处理12周的OVX小鼠的代表性μCT图像（ 图1B ，右图）。在EXD治疗的小鼠的腰椎椎弓?…
Discussion
近年来，更多的关注草药，一种在东方世界临床环境中应用了数千年的替代药物。传统草药与现代医学的“长凳式”床不同，首先需要“床到凳”的模式来解释他们的机制。这可以在后台进行验证，并进行开发新优化药物的处理。到目前为止，已经报道了从整个配方或草药释放活性成分的几种提取方法。其中，水提取是最广泛使用的。
中草药的提取有四个基本步骤。第一步?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国国家自然科学基金（81573992）的支持。我们感谢Emily K. Lo和Kathleen DiNapoli的语言编辑协助。
Materials
| Curculigo orchioides Gaertn (9 g), Herbaa Epimedii (9 g), Radix Morindae Officinalis (9 g), Radix Angelicae Sinensis (9 g), Cortex Phellodendri (6 g), and Rhizoma Anemarrhenae (6 g) | Kang-qiao Chinese Medicine Yinpian Co. Ltd (Shanghai, CN) | 160922 | EXD components |
| Filter paper | GElifesciences | 99-103-952 | Filter EXD decoction before concentration |
| Imprinting Control Region (ICR) mice | Shanghai Laboratory Animal Center | SCXK 2007-0005 | In vivo study |
| Sprague Dawley rats | Shanghai Laboratory Animal Center | SCXK 2007-0005 | EXD-containing serum preparation |
| Syringe filter | Millipore | SLGP033RB | 0.22 µm |
| gavage needles (10 ml) | Shanghai BO Ge trade sales department | 59104274 | Adminstration of EXD |
| Ketamine (80 mg/kg) | Fujian Gutian Pharma Co. Ltd | H35020148 | Anesthesia |
| Xylazine (10 mg/kg) | Sunway Pharma Co. Ltd | CB07591 | Anesthesia |
| Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM) culture medium | Gibco | 12800-116 | DMEM with 2 mM L-glutamine and without ribonucleosides and ribonucleotides |
| Streptomycin | Sigma | 1277 | 100 µg / ml |
| Penicillin | Sigma | 4687 | 100 µg / ml |
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Citer Cet Article
Liu, S., Sun, Y., Li, J., Dong, J., Bian, Q. Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments. J. Vis. Exp. (123), e55654, doi:10.3791/55654 (2017).