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。

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 <ol><li> 计算 <ol><li>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。 </li><li>计算每只小鼠施用的EXD的体积（ 即 V =（0.1418EXD / kg·天）*（0.02kg /小鼠）* 50mL / EXD = 0.14mL /小鼠·天）。 </li></ol></li><li> 浸水 <ol><li>将两种EXD（ 即，Curculigo orchioides Gaertn （18 g）， Herbaa Epimedii （18 g））， 甘草 （18 g）， R将当归（18g）， 皮质黄柏 （12g）和黑曲霉 （12g），总共96g）放入具有盖子的容器中。 注意:建议使用陶瓷容器。 </li><li>用500毫升蒸馏水将原始草药切片1小时;水应该覆盖草药约一英寸。让所有的草药彻底浸泡。 </li></ol></li><li> 第一水煎剂 <ol><li>用大功率的燃气灶或电磁炉加热草药，直到水沸腾（约5-10分钟;时间间隔取决于加热功率，容器和草药和水量）。将电源降低到低炖煮2小时。 </li></ol></li><li> 先过滤 <ol><li>用常规滤纸或纱布和棉布覆盖500 mL玻璃烧杯。通过过滤器小心倒入烧杯中。把草药放在容器中。 </li><li>返还剩余的草药残留物o把滤纸放到容器上。 </li></ol></li><li> 第二汤水 <ol><li>用草药将蒸馏水加入容器中;让它覆盖草药约一英寸。 </li><li>重复步骤1.3。 </li></ol></li><li> 二次过滤 <ol><li>重复步骤1.4.1和1.4.2。 </li><li>将第二汤匙倒入同一个烧杯中。将第一和第二汤匙混合在一起。 </li></ol></li><li> 浓度 <ol><li>将烧杯放在燃气灶之间，用无石棉丝网将它们之间加热，用低温炖煮，并用玻璃棒慢慢地连续搅拌。 </li><li>当煎剂减少到200 mL时，将其转移到500 mL烧杯中。 </li><li>重复步骤1.7.1，直到煎剂减至100 mL。 </li></ol></li><li> 储存和管理 <ol><li>将浓缩汤汁转移到无菌玻璃瓶中。让它凉快到室温re（RT）。如果在一周内或-70°C使用长期储存，请在4°C储存。 </li><li>使用管饲针，向卵巢切除（OVX）小鼠施用0.14mL /小鼠煎剂一天，每周5天，持续12周。 </li></ol></li></ol> 2.议定书二:含EXD的血清的制备<ol><li> 计算 <ol><li>计算每只大鼠（1个月大）体重0.15 kg的大鼠当量（RED）:dB =（1/60）*（90/100）*（60 / 0.15） 1/3 = 0.111 EXD / kg /天。 注意:需要总共10ml的含EXD血清以制备100mL培养基（含10％含EXD的血清）。预计每只大鼠提供2mL的血清。因此，每天一次，需要6只大鼠（20％的损失被考虑在内）3天。 EXD的数量是:（0.111EXD / kg•天）*（0.15kg /大鼠）*（6只大鼠）*（3天）= 0.300EXD。再次，因为EXD以离散增量测量，使用1 EXD。 </li><li>计算每只大鼠施用的EXD体积（ 即 V =（0.111EXD / kg·天）*（0.15kg /大鼠）*（50mL / EXD）= 0.83mL /大鼠·天）。 </li></ol></li><li> 浸水 <ol><li>将1 EXD的原料放入带盖的容器中。将原料在蒸馏水中浸泡1小时;水应该覆盖草药约一英寸。让所有的草药彻底浸泡。 </li></ol></li><li> 第一水煎剂 <ol><li>用高功率的电磁炉加热草药，直到水沸腾（约5 - 10分钟;时间间隔取决于热量，容器和草药和水量）。将电源切换到低炖煮2小时。 </li></ol></li><li> 先过滤 <ol><li>用常规滤纸或纱布和棉布覆盖500 mL玻璃烧杯。通过过滤器小心倒入烧杯中。把草药放在容器中。 </li><li&gt;将滤纸上的草药残渣送回容器。 </li></ol></li><li> 第二汤水 <ol><li>用草药将蒸馏水加入容器中;让它覆盖草药约一英寸。 </li><li>重复步骤2.4。 </li></ol></li><li> 二次过滤 <ol><li>重复步骤2.5.1和2.5.2。 </li><li>将第二汤匙倒入与步骤2.4.1中相同的烧杯中，并将第一和第二汤匙混合在一起。 </li></ol></li><li> 浓度 <ol><li>将烧杯放在燃气灶之间，用无石棉丝网将它们之间加热，并用低温炖煮，并用玻璃棒慢慢连续搅拌。 </li><li>当煎剂降至100 mL时，将其转移到100 mL烧杯中。 </li><li>重复步骤2.7.1，直到煎煮至50 mL。 </li></ol></li><li> 储存和管理 <ol><li>将浓缩汤汁转移至灭菌剂中ile玻璃瓶。让它凉爽到RT。如果在一周内或-70°C使用长期储存，请在4°C储存。 </li><li>腹腔内给予0.83mL /天，每天一次，持续3天。 </li></ol></li><li> 含EXD的血清制剂 <ol><li>在最后一次给药后1小时腹腔注射300 mL / 100g 80 mg / kg氯胺酮和10mg / kg甲苯噻嗪麻醉麻醉大鼠。通过脚趾捏捏确认适当的麻醉。 </li><li>使用手术刀（或直接操作的剪刀）将大鼠的皮肤和腹膜从腹部切除到胸部的底部。创建长度和深度分别为5厘米和0.5厘米的切口。使用薄纸将腹部内脏移至左侧。 </li><li>使用薄纸，去除腹主动脉的结缔组织，以清楚地暴露血管。 </li><li>使用10 mL，22号注射器从腹主动脉缓慢吸取血液。转移到取出针后取出15 mL无菌管;一只大鼠可以产生8-10毫升血液。 注意:开始抽血时，大鼠应该活着（ 即腹部主动脉脉动），完成后，死亡。 </li><li>在室温下将血液凝固在直立位置30-60分钟。以500-600 xg离心20分钟。将所有上清液（血清）小心地放入一个50mL无菌管中，并将所有血清（来自不同动物）混合在一起。 </li><li>通过在56℃水浴中孵育血清30分钟来进行热灭活。使用具有0.22μm孔径的亲水性聚醚砜膜的注射器过滤器过滤血清。使用新鲜血清或将其储存在-20°C。 </li></ol></li><li> 对照药物血清制剂（用于对照组） <ol><li>用相同体积的盐水（0.83mL）每天施用每只大鼠一次，持续3天。其他步骤与步骤2.9相同。</li></ol></li><li> 应用 <ol><li>向每100mL培养基加上1％的青霉素 - 链霉素（PS） 23加入含有EXD的血清或含药血清。 </li></ol></li></ol>

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作者宣称他们没有竞争的经济利益。

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。第一步是浸泡。复杂的草药在水中浸泡一段时间，通常为0.5至1小时。浸泡时间取决于原料的数量和性质。至确保原料彻底浸泡，每片或块的中心应浸泡。水的体积取决于草药的总体积。有时，草药的重量用于确定浸泡需要多少水。然而，草药密度差异很大。由于浸泡的目的是浸渍所有草药，因此在以下步骤中促进活性组分的释放，草药的体积优选用作水体积的参考标准。第二步是做饭。煮沸和煨煮的过程已经发展了数千年。烹饪时间的变化主要取决于草药的种类，因为它们的物理，化学和药理特性不同12 。建议将diaphoretics短时间内烹饪，通常少于20分钟，而在第一次煮沸前只应加入芳香剂几分钟，以避免挥发性反应。 F或补药，需要很长时间才能浸出其治疗成分。在这种情况下，EXD用于治疗绝经后骨质疏松症几十年。在中医理论中，这种综合征被认为是由肾相关的缺乏引起的。 EXD主要用于补肾。因此，EXD煮熟2 - 3小时，让EXD发挥其抗骨质疏松作用15 。第三步是过滤。过滤有助于防止管饲针被草药颗粒堵塞。第四步是集中。应慎重考虑管饲管理量。据报道，通过口服管饲法施用的大量体积（例如10mL / kg或更多）可导致与吸收相关的若干问题，包括将化合物快速分流至十二指肠或与被动反流相关的吸入性肺炎本草进入食道25 。因此，为小鼠和大鼠选择的体积分别为约7.5mL / kg和5.5mL / kg。 重要的是确认血液中消化和代谢的EXD的有效成分是否与原始提取物相似。在EXD生物等效性方面，Wu et al。报道了狗血浆中7种成分的测定:依马西林A，依马西林B，依马西林C，淫羊藿苷，箭头糖苷B，2"-O-鼠李糖苷II和宝果苷I 26 。胡等口服后大鼠血浆中发现21种化合物27 。迄今为止，EXD的原始提取物尚未报道。然而，在血液中测试的一些有效成分，如淫羊藿苷和小檗碱已被直接应用于动物进行比较观察28 。 有多种方法来计算动物生物等价剂量。基于体重（mg / kg）的传统方法是不合适的，因为不同物种的药代动力学变化不同。经常使用基于身体表面积（mg / m 2 ）的计算，其中代谢率与个体动物大小有关。这里应用的方程式考虑了体表面积和体重，在中医药中普遍使用30 。 可以选择许多种类的动物，例如兔，豚鼠，大鼠和小鼠，用于含药血清制剂。与体外处理的细胞相同的物种是优选的。在这项研究中，选择大鼠，因为它们比小鼠提供更多的血清，并且与其他动物相比，它们与物种相比更接近于小鼠。此外，推荐体内或临床使用的剂量当量，如体外方案所示。稀释不考虑血清离子（推荐1:10）也就是说，由于由处理的细胞或器官引起的潜在的毒性反应，等效剂量的10倍不适用于血清提供的动物。药物管理频率每天一次为3至14天，每天两次（每次给药之间2小时）为7,8,32。收集时间通常在最后一次管理33,34之后的小时1和2（6小时之前）之间发生。目的是在采集样品时，将血液中的药物浓度保持在相对稳定和峰值水平。根据体内给药程序，给药程序可以包括注射，皮肤给药或吸入。 药物 -含血清仍有争议。支持者认为许多活性成分的存在，如激素，酶，抗体和血清本身的补体可能会导致意想不到的反应，影响结果36 。反对派认为，药物产生的活性成分也可能被灭活过程37除去。为了平衡这一点，人们设计对照组，使用来自盐水处理的动物的血清。 协议有一些限制。在开始体内和体外实验之前，不评估提取物的质量。其次，在培养基中使用抗生素，这可能导致草药相互作用，需要进一步的检测。第三，浸泡和煨时间是根据临床实践和动物实验获得的经验来确定的。更多的时间可以选择f或比较。浸泡和煨时间可以修改为不同的草药配方。可以根据动物的种类，年龄和体重来改变动物给药的煎煮体积。选择用于制备含药血清和用于给药程序的动物可以改变，如上所述。 总而言之，方案和结果为在体内和体外研究中草药煎剂的制备和应用提供了一个例子。在不同的情况下，一些细节必须优化，包括治疗期，物种和管理程序，基于草药特征。

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。 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 + ， 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）。

<table><tbody><tr><td>Curculigo orchioides Gaertn (9&amp;thinsp;g), Herbaa Epimedii (9&amp;thinsp;g), Radix Morindae Officinalis (9&amp;thinsp;g), Radix Angelicae Sinensis (9&amp;thinsp;g), Cortex Phellodendri (6&amp;thinsp;g), and Rhizoma Anemarrhenae (6&amp;thinsp;g)</td><td>Kang-qiao Chinese Medicine Yinpian Co. Ltd (Shanghai, CN)</td><td>160922</td><td>EXD components</td></tr><tr><td>Filter paper</td><td>GElifesciences</td><td>99-103-952</td><td>Filter EXD decoction before concentration</td></tr><tr><td>Imprinting Control Region (ICR) mice</td><td>Shanghai Laboratory Animal Center</td><td>SCXK 2007-0005</td><td>In vivo study</td></tr><tr><td>Sprague Dawley rats</td><td>Shanghai Laboratory Animal Center</td><td>SCXK 2007-0005</td><td>EXD-containing serum preparation</td></tr><tr><td>Syringe filter</td><td>Millipore</td><td>SLGP033RB</td><td>0.22 &amp;micro;m</td></tr><tr><td>gavage needles (10 ml)</td><td>Shanghai BO Ge trade sales department</td><td>59104274</td><td>Adminstration of EXD</td></tr><tr><td>Ketamine (80 mg/kg)&amp;nbsp;</td><td>Fujian Gutian Pharma Co. Ltd</td><td>H35020148</td><td>Anesthesia</td></tr><tr><td>Xylazine (10 mg/kg)</td><td>Sunway Pharma Co. Ltd</td><td>CB07591</td><td>Anesthesia</td></tr><tr><td>Dulbecco&amp;rsquo;s modification of Eagle&amp;rsquo;s medium Dulbecco (DMEM) culture medium</td><td>Gibco</td><td>12800-116</td><td>DMEM with 2 mM L-glutamine and without ribonucleosides and ribonucleotides</td></tr><tr><td>Streptomycin</td><td>Sigma</td><td>1277</td><td>100 &amp;micro;g / ml</td></tr><tr><td>Penicillin</td><td>Sigma</td><td>4687</td><td>100 &amp;micro;g / ml</td></tr></tbody></table>

preparation of herbal medicine: er-xian decoction and er-xian-containing serum for in vivo and in vitro experiments

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。

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根内观察到的小骨小于对照OVX小鼠。来自μCT成像的数据表明第4腰椎的骨量/组织体积（BV / TV），小梁数（Tb.N）和小梁厚度（Tb Th）和骨小梁间距（Tb.Sp）减小<html>在EXD小鼠中（ 图1C ）。 EXD对来自OVX小鼠的骨间充质干细胞（bMSCs）的骨形成的影响 图2表示骨髓基质干细胞的成骨潜力。 OVX小鼠的骨髓基质干细胞培养7天后，自动形成脂滴（星号，脂滴的不规则形状）（ 图2A ，左图）。在EXD处理的小鼠中，形成骨结节（箭头）代替脂肪液滴（ 图2B ，右图）。碱性磷酸酶（ALP）测定显示与来自对照OVX小鼠的bMSCs相比，在EXD处理的骨髓基质干细胞（ 图2B ，右图）中可以检测到更多的ALP阳性细胞（紫色）（ 图2B ，左图）。 OVX和EXD小鼠之间基因表达的变化S = "外部参照"&gt; 24 分层聚类表明，OVX和EXD处理的小鼠体内的 bMSCs中微阵列揭示的389个基因的表达变化（大于1.5，非OVX小鼠标准化）（总共26,991个基因）（ 图3 ）。绿色表示表达式被上调，而红色表示表达式被下调。同一组中的三个样品首先聚集在一起，表明轮廓的可靠性质。 图4显示了体内和体外 EXD靶向的重叠信号传导途径。 <img alt="图1" src="/files/ftp_upload/55654/55654fig1.jpg" /> 图1:EXD对骨形态的影响。 （ A ）OVX和EXD处理的小鼠的腰椎第 4部分的H＆E染色。 （ <strB）对照OVX和EXD处理的小鼠中腰椎第 4小梁骨的三维μCT重建图像。 （ C ）μCT的定量。 BV:骨量，TV:组织体积，Tb.N:小梁数，Tb.Th:小梁厚度，Tb.Sp:小梁间距。列表示平均值±SE。每组n = 6。 * p &lt;0.05，** p &lt;0.01 EXD与OVX（学生t检验）。 （ AC ）已从Shufen Liu 等人修改<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55654/55654fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55654/55654fig2.jpg" /> 图2:EXD对bMSC分化的影响。 （ A ）biC培养7天后的图像从OVX或EXD处理的小鼠的股骨中分离出来。 *表示脂肪液滴。箭头表示骨结节。 （ B ）培养7天的OVX和EXD处理的骨髓基质干细胞的ALP染色（紫色代表ALP阳性）。 （ C ）（ B ）的定量。列表示每组三个皿（六只小鼠）的平均值±SE。 ** p &lt;0.01 EXD与OVX（学生t检验）。 （ AC ）已从Shufen Liu 等人修改<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55654/55654fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55654/55654fig3.jpg" /> 图3:EXD对基因表达谱的影响。 OVX和EXD骨髓基质干细胞中389个基因表达分层聚类的热图<html>在分离后第七天收获。比例（小图像）表示由非OVX骨髓基质干细胞标准化的倍数变化。基因列表在补充文件1中提供。该图已经从Shufen Liu 等人修改<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55654/55654fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" src="/files/ftp_upload/55654/55654fig4.jpg" /> 图4:EXD对体内和体外实验中重叠信号通路的影响。 基于KEGG途径体外 EXD 体外和EXD的血清逆转的前10个重叠信号通路。与通路相关的基因见补充文件2 。/files/ftp_upload/55654/55654fig4large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 种类 </td><td> 老鼠 </td><td> 鼠 </td><td> 豚鼠 </td><td> 兔子 </td><td> 猫 </td><td> 猴 </td><td> 狗 </td><td> 人的 </td></tr><tr><td> [R </td><td> 59 </td><td> 90 </td><td> 99 </td><td> 93 </td><td> 82 </td><td> 111 </td><td> 104 </td><td> 100 </td></tr></tbody></table> 表1:不同物种的R因子。 8种通常用于药理研究的R因子。 R因子与（体表面积（m 2 ）/ b成比例）重量（kg）） 2/3 。 补充文件1。 关于389个上调或下调≥1.5倍但由体外 EXD拯救的基因的信息。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55654/Supplemental_file_1.xls" target="_blank">请点击这里下载此文件。</a> 补充文件2。 关于信号通路（基于KEGG途径）和相关基因在体内和体外实验中的信息。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55654/Supplemental_file_2.xls" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments at JoVE.com

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

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。

草药的制备:洱西汤和含茜素的血清<em&gt;体内</em&gt;和<em&gt;体外</em&gt;实验

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the ...

preparation-herbal-medicine-er-xian-decoction-er-xian-containing

Universidad San Sebastian

Research

JoVE Journal

Medicine

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

10.9K 次观看.  Shanghai University of Traditional Chinese Medicine. 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。 

视频: Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United States (US) and Europe. Berries are widely consumed in the US and have a high polyphenolic content. Polyphenols have been shown to interact with many molecular targets and to exert numerous positive biological functions, including antioxidant, anti-inflammatory, and cardioprotective effects. Polyphenols isolated from blackberry (BL), raspberry (RB), and black raspberry (BRB) reduce oxidative stress and cellular senescence in response to angiotensin II (Ang II). This work provides a detailed description of the protocol used to prepare the polyphenol extracts from freeze-dried berries. Polyphenol extractions from freeze-dried berry powder were performed using 80% aqueous ethanol and an ultrasonic-assisted extraction method. The crude extract was further purified and fractionated using chloroform and ethyl acetate, respectively. The effects of both crude and purified extracts were tested on Vascular Smooth Muscle Cells (VSMCs) in culture.

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

This work details a step-by-step method to prepare polyphenol-rich extracts from freeze-dried berry powder. In addition, it provides a thorough description of how to use these polyphenol-rich extracts in cell culture in the presence of the peptide hormone angiotensin II (Ang II) using Vascular Smooth Muscle Cells (VSMCs).

从冷冻干燥的浆果粉中提取和纯化多酚用于治疗血管平滑肌细胞<em&gt;体外</em

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United ...

科研

化学

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.

<meta charset="utf8"></head><body>超高效液相色谱-质谱法及自建中草药成分数据库分析

We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal medicine.

超高效液相色谱-质谱法及自建中草药成分数据库分析

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

古树康 (GSK) 颗粒的制备,用于 Vivo 和体外实验

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

医学

Preparation of Naringenin Solution for In Vivo Application

The preparation of a compound (phytochemical) solution is an overlooked but critical step prior to its application in studies such as drug screening. The complete solubilization of the compound is necessary for its safe use and relatively stable results. Here, a protocol for preparing naringenin solution and its intraperitoneal administration in a high-fat diet and streptozotocin (STZ)-induced diabetic model is demonstrated as an example. A small amount of naringenin (3.52-6.69 mg) was used to test its solubilization in solvents, including ethanol, dimethylsulfoxide (DMSO), and DMSO plus Tween 80 reconstituted in physiological saline (PS), respectively. Complete solubilization of the compound is determined by observing the color of the solution, the presence of precipitates after centrifugation (2000 x g for 30 s), or allowing the solution to stand for 2 h at room temperature (RT). After obtaining a stable compound/phytochemical solution, the final concentration/amount of the compound required for in vivo studies can be prepared in a solvent-only (no PS) stock solution, and then diluted/mixed with PS as desired. The antidiabetic osteoporotic effects of naringenin in mice (intraperitoneal administration at 20 mg/kg b.w., 2 mg/mL) were assessed by measuring blood glucose, bone mass (micro-CT), and bone resorption rate (TRAP staining and ELISA). Researchers looking for detailed organic/phytochemical solution preparations will benefit from this technique.

Preparation of Naringenin Solution for In Vivo Application

Here, the protocol presents the preparation of naringenin solution for in vivo intraperitoneal administration. Naringenin is fully dissolved in a mixture of dimethylsulfoxide, Tween 80, and saline. The antidiabetic osteoporotic effects of naringenin were assessed by blood glucose testing, tartrate-resistant acid phosphatase staining, and enzyme-linked immunosorbent assay.

柚皮素溶液的制备

The preparation of a compound (phytochemical) solution is an overlooked but critical step prior to its application in studies such as drug screening. The ...

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, establishing a method to determine the multi-component in vitro dissolution of RG is of great significance for quality control. This study uses the second paddle method of the fourth general rule 0931 from the Chinese Pharmacopoeia (2020 edition), compliant with apparatus 2 of the United States Pharmacopeia (USP). The dissolution apparatus was set to a rotation speed of 100 rpm with ultrapure water as the dissolution medium. A sample volume of 1 mL was collected at each timepoint. Furthermore, the cumulative dissolution of gallic acid, salidroside, and ethyl gallic acid in RG at different time points was determined by high-performance liquid chromatography (HPLC). Finally, the dissolution curves were drawn, and the curves were fitted to the GompertzMod, the Gompertz, the Logistic, and the Weibull equations. The results showed that the cumulative dissolution of gallic acid in RG was over 80% at 1 min, the cumulative dissolution of salidroside and ethyl gallic acid was over 65% at 5 min, and the cumulative dissolution of each index component decreased after 30 min. The curve fitting demonstrated that the GompertzMod equation was the best-fitting model for each index component of RG. In conclusion, the dissolution test method described in this protocol is simple, accurate, and reliable. It can characterize the dissolution behavior of the index components in RG in vitro, which provides a methodological reference for quality control of RG and quality evaluation of other ethnic compounds.

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

Here, we test the dissolution of Rhodiola granules (RG) in vitro, draw dissolution curves of salidroside, gallic acid, and ethyl gallate in ultrapure water, and fit the curves to different mathematical models. This protocol provides information and guidance for in vivo bioequivalence and in vivo-in vitro correlation studies of RG.

藏药红景天颗粒中多指标组分的 体外 溶出度测定

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, ...

Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in the efficacy and mechanism between the preparation combination and the component combination in order to explore the two herb combination strategy to treat injured PC12 cells. Cobalt chloride (CoCl2) combined with a glucose-free medium was employed to induce oxidative damage of PC12 cells. Then, the optimal combination of Astragalus mongholicus (Ast) and Erigeron breviscapus (Eri) injection was selected and combined following uniform design methods after screening their safe and effective concentration on PC12 cells. Further, the component combination screened comprises 10 &#181;M astragaloside A, 40 &#181;M scutellarin, and 75 &#181;M chlorogenic acid in two herbs. Then, MTT, Annexin V-FITC/PI, immunofluorescence, and Western blot analysis were used to evaluate the efficacy and mechanism of the preparation combination and the component combination on injured PC12 cells. The results showed that the optimal preparation combination for cell pro-survival was Ast injection and Eri capsule with a concentration of 6:1.8 (&#181;M). The component combination (10 &#181;M astragaloside A, 40 &#181;M scutellarin, and 75 &#181;M chlorogenic acid) was more effective than the preparation combination. Both combinations remarkably reduced apoptotic rate, the fluorescence intensity of caspase-3, and intracellular reactive oxygen species (ROS) level; meanwhile, they upregulated the expression levels of p-Akt/Akt, Bcl-2/Bax, and Nrf2. These effects were more evident in the component combination. In conclusion, both combinations can inhibit the injury induced by CoCl2 combined with a glucose-free medium on PC12 cells, thus promoting cell survival. However, the efficiency of the component combination over the preparation combination may be due to its stronger regulation of the PI3K/Akt/Nrf2 signaling pathway related to oxidative stress and apoptosis.

This protocol provides a combination strategy of two herbs to treat injured PC12 cells. The protocol provides a reference for optimizing the best application mode of traditional Chinese medicine (TCM).

探索两种草药组合策略治疗受损PC12细胞

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in ...

Optimization of the Epimedii Folium Mutton-Oil Processing Technology and Testing Its Effect on Zebrafish Embryonic Development

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 with mutton oil is often used as a medicine. In recent years, reports of safety risks and adverse reactions of products that use EF as a raw material have gradually increased. Processing can effectively improve the safety of TCM. According to TCM theory, mutton-oil processing can reduce the toxicity of EF and enhance its tonifying effect on the kidneys. However, there is a lack of systematic research and evaluation of EF mutton-oil processing technology. In this study, we used the Box-Behnken experimental design-response surface methodology to optimize the key parameters of the processing technology by assessing the contents of multiple components. The results showed that the optimal mutton-oil processing technology of EF was as follows: heating the mutton oil at 120 &#176;C &#177; 10 &#176;C, adding the crude EF, stir-frying it gently to 189 &#176;C &#177; 10 &#176;C until it is evenly shiny, and then removing it and cool. For every 100 kg of EF, 15 kg of mutton oil should be used. The toxicities and teratogenicities of an aqueous extract of crude and mutton-oil processed EF were compared in a zebrafish embryo developmental model. The results showed that the crude herb group was more likely to cause zebrafish deformities, and its half-maximal lethal EF concentration was lower. In conclusion, the optimized mutton-oil processing technology was stable and reliable, with good repeatability. At a certain dose, the aqueous extract of EF was toxic to the development of zebrafish embryos, and the toxicity was stronger for the crude drug than for the processed drug. The results showed that mutton-oil processing reduced the toxicity of crude EF. These findings can be used to improve the quality, uniformity, and clinical safety of mutton oil-processed EF.

In this protocol, the mutton-oil processing technology of Epimedii folium (EF) was optimized by applying a Box-Behnken experimental design-response surface methodology, and the effect of crude and optimized water-extracted EF on zebrafish embryonic development was preliminarily investigated.

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 ...

<meta charset="utf8"></head><body>三棱佳申方对小鼠原位肝细胞癌的影响

Establishment of Hepatocarcinoma in BALB/c-nu Mice and Investigation of the Therapeutic Effect of the Sanleng Jiashen Formula

The lethality of liver cancer and the resistance to chemical drugs have forced the search for effective prescriptions of traditional herbs for liver cancer. Animal models that are repeatable, easy to manipulate, and highly mimic the pathophysiological processes of liver cancer are the prerequisite for the successful screening of effective drug candidates. Meanwhile, reliable drug efficacy evaluation indicators and means are also the guarantee of anti-liver cancer drug research and development.
Sanleng Jiashen formula, a representative prescription of traditional Chinese medicine containing Sparganium stoloni erum, Buch. -Ham. (Sanleng), Panax ginseng C. A. Mey. (ginseng), Rheum officinale Baill. (rhubarb), and Ligusticum chuanxiong Hort. (Chuanxiong), is prescribed to nourish the liver and clear heat, remove toxins, and promote blood circulation to treat liver cancer. This experimental protocol describes the preparation of lyophilized Sanleng Jiashen formula and the establishment process of in-situ liver cancer in BALB/c-nu mice. Histopathological staining, immunohistochemical detection of cancer markers, in vivo imaging of mice, and chick embryo chorioallantoic membrane test were used to explore the inhibition and anti-angiogenesis effect of Sanleng Jiashen formula on malignant proliferation of liver cancer tissue. The data show that the Sanleng Jiashen formula can effectively resist the malignant proliferation of liver cancer tissue, which is manifested by reduced tumor mass volume, improved pathological damage, and lower levels of the cancer marker ki67.
The superior inhibition of angiogenesis also suggests that the Sanleng Jiashen formula may have the potential to treat and prevent the progression and deterioration of liver cancer. The whole experimental scheme shows a comprehensive process of traditional Chinese medicine components in the treatment of mouse liver cancer, which provides a reference for the establishment and optimization of a liver cancer model, as well as the research and development of drugs to prevent and treat liver cancer.

<meta charset="utf8"></head><body>BALB/c-nu 小鼠肝癌的建立及三棱家参方治疗效果的探讨

Here, we present the step-by-step protocol to investigate the therapeutic and vascular inhibitory effects of the Sanleng Jiashen formula on BALB/c-nu mice with orthotopic hepatocellular carcinoma.

BALB/c-nu 小鼠肝癌的建立及三棱家参方治疗效果的探讨

The lethality of liver cancer and the resistance to chemical drugs have forced the search for effective prescriptions of traditional herbs for liver ...

草药的制备:洱西汤和含茜素的血清

摘要

探索更多视频

从冷冻干燥的浆果粉中提取和纯化多酚用于治疗血管平滑肌细胞

超高效液相色谱-质谱法及自建中草药成分数据库分析

超高效液相色谱-质谱法及自建中草药成分数据库分析

古树康 (GSK) 颗粒的制备,用于 Vivo 和体外实验

柚皮素溶液的制备

藏药红景天颗粒中多指标组分的体外溶出度测定

探索两种草药组合策略治疗受损PC12细胞

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

BALB/c-nu 小鼠肝癌的建立及三棱家参方治疗效果的探讨

BALB/c-nu 小鼠肝癌的建立及三棱家参方治疗效果的探讨

草药的制备:洱西汤和含茜素的血清

摘要

探索更多视频

从冷冻干燥的浆果粉中提取和纯化多酚用于治疗血管平滑肌细胞

超高效液相色谱-质谱法及自建中草药成分数据库分析

超高效液相色谱-质谱法及自建中草药成分数据库分析

古树康 (GSK) 颗粒的制备,用于 Vivo 和体外实验

柚皮素溶液的制备

藏药红景天颗粒中多指标组分的 体外 溶出度测定

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