本研究项目由（1）国家自然科学基金（81973877 82174408）、（2）国家重点研发计划（2020YFE0201600）、（3）上海市重点研究中心建设项目（2022ZZ01009）、（4）上海市医院中药制剂产业转化协同创新中心、（5）上海中医药大学预算内研究项目（2021LK047）资助。

本方案以益奇节度狙为样本，描述了骨肉瘤模型小鼠的详细管饲技术。本实验采用4周龄雄性无胸腺小鼠（BALB/c裸体），饲养在上海中医药大学实验动物中心SPF级育种室。所有动物实验均经上海中医药大学动物管理委员会批准，符合《实验动物伦理规范》（动物伦理批准号：PZSHUTCM2304060007），严格按照国际实验动物相关要求进行。
1. 材料
<ol><li>获取以下材料：中草药（人参 42 g、 Hedyotis diffusa 52.5 g、带刺黄芩草本 31.5 g）、1 mL 注射器和 12G/55 mm 肘部管饲针（图 1）。</li>
</ol>2.剂量测定
<ol><li>按以下方式计算日用量：标准体重60kg的成虫日用量为人参12g、 弥漫蓿 15g、倒刺黄芩草9g，共36g即饮药，即0.6g即食药/kg体重。参考标准体重60kg的成虫与标准体重25g的小鼠11的等效剂量12倍、低剂量6倍、高剂量24倍，以及我们的初步数据，本研究中对小鼠使用剂量比例为20倍的益齐节度汤， 因此，小鼠的剂量为12g即食药物/kg体重。</li>
	<li>使用以下公式计算每只小鼠的剂量： 小鼠剂量（站立体重）=成人剂量（站立体重）×20（指定等效剂量比）。 每只小鼠的日剂量为0.3g[（12g即食药物/ 1000g体重）x 25g体重]在200μL中，终浓度为1.5g/ mL。</li>
	<li>计算强饲给药28天的益琦节度汤组小鼠（n=7）的总煎药为58.8g。将其浓缩至 39.2 mL，以达到 1.5 g/mL 的最终浓度。</li>
	<li>考虑到煎煮和管饲阶段可能发生的药物损失，为了方便计算，制备共126g（人参 42g，Hedyotis diffusa 52.5g，带刺黄芩草31.5g）煎药，浓缩成最终体积为84mL。</li>
</ol>3. 细胞来源和培养
<ol><li>从ATCC细胞库购买人源性143B骨肉瘤细胞。</li>
	<li>在 10 cm 细胞培养皿中将 1 x 106 143B 细胞接种在含有 10% FBS、100 U/mL 青霉素和 100 μg/mL 链霉素的 10 mL MEM 培养基中。</li>
	<li>在37°C下在5%CO2 培养箱中培养143B细胞，直至细胞80%-90%汇合。</li>
</ol>4. 建模方法
<ol><li>收集 80% 汇合培养的 143B 细胞，重悬于 6 mL 含 10% 血清的 MEM 培养基中，并以 1：1 的体积比与基底膜基质凝胶混合，制成终浓度为 1 x 107 个细胞/mL 的细胞悬浮液。</li>
	<li>将 10 μL 细胞悬液注射到每只无胸腺小鼠的左胫骨中，以建立 原位 异种移植骨肉瘤小鼠模型，如我们之前的出版物12 中所述。步骤简述如下。<ol><li>用PBS洗涤细胞2次。用 1.5 mL 0.25% 胰蛋白酶使细胞胰蛋白酶消化 3 分钟。加入 6 mL 含 10% 血清的 MEM 培养基。将细胞收集在 15 mL 离心管中。</li>
		<li>将 20 μL 细胞悬液吸入细胞计数板的腔室中，并使用自动细胞计数器计算细胞浓度。在室温下以800× g 离心细胞5分钟。</li>
		<li>用移液管弃去上清液，将细胞沉淀重悬于8.5mg / mL基底膜基质中，并将其置于冰上。</li>
		<li>通过将小鼠暴露于2%异氟烷和98%氧气（氧流速，2L / min）来麻醉小鼠。在眼睛上涂抹少量兽医软膏，以防止麻醉时干燥。</li>
		<li>在通风良好的地方进行整个过程。在骨肉瘤细胞注射前，通过轻轻触摸脚部，确保每只小鼠处于深度麻醉状态;如果鼠标仍有反应，例如抽搐或抽搐。如果鼠标仍有反应（如抽搐或抽搐），请等待更长的时间，直到这些反应消失。</li>
		<li>将鼠标置于仰卧位。用拇指和食指握住鼠标的脚踝，并用三轮交替的甜菜碱和70%乙醇拭子对胫骨注射部位进行消毒。</li>
		<li>将针头连接到 1 mL 注射器上，并将针尖指向平行于胫骨轴线的注射部位。经皮插入针头，在胫骨远端钻一个孔，穿过胫骨平台。</li>
		<li>将先前放在冰上的骨肉瘤细胞悬液加载到微量注射器中，并用载有骨肉瘤细胞的微量注射器替换胫骨中的 1 mL 注射器。缓慢将约10μL（1×10,7个细胞/ mL）骨肉瘤细胞悬液注射到每个无胸腺小鼠的胫骨中。</li>
		<li>从注射部位取出微量注射器，用棉签按压注射部位约30秒。将鼠标放回干净的笼子中，观察其恢复10分钟。</li>
	</ol></li>
</ol>5.中药管饲配方的制备（图2）
<ol><li>按照步骤2计算剂量。</li>
	<li>将除昂贵的药材人参外的所有药材浸泡在比药材表面高3厘米的饮用水中30分钟，然后如下所述进行2次煎煮。</li>
	<li>通过在强火上加热至沸腾，然后在温和的火上加热15-25分钟来进行第一次煎剂。轻度火灾是指温度通常在 80 °C 至 100 °C 之间的低功率热量，而强火是指通常在 200 °C 至 300 °C 之间的高功率热量。</li>
	<li>将汤液倒入一个容器中。</li>
	<li>通过在饮用水中加热药材进行第二次煎剂，其体积刚好足以淹没药物表面，在强火上煮沸，然后在温和的火上加热15-25分钟。</li>
	<li>将煎剂倒入与步骤5.4相同的容器中。</li>
	<li>将42克人参分别浸泡和煎煮，饮用水比药面高3厘米，因为它很贵。</li>
	<li>通过在强火上加热60分钟来执行浸泡过的人参的第一次煎煮。 将煎剂倒入与步骤5.4相同的容器中。</li>
	<li>在饮用水中进行人参的第二次煎煮，其体积刚好足以淹没药物表面，在温和的火上加热40分钟。将煎剂倒入与步骤5.4相同的容器中。 注意：用于煎剂的容器应具有化学稳定性，例如砂锅菜（临床实践中最常用的）、陶瓷、玻璃和不锈钢，以避免与药物发生化学反应。所有从不同步骤获得的汤液必须放入同一容器中。</li>
	<li>使用用橡皮筋固定在量杯口的三层纱布混合和过滤汤液。</li>
	<li>将过滤后的汤剂在锅中用强火煮沸，然后换成温和的火，保持温和的火，直到达到目标浓度（终体积为84mL，终浓度为1.5g/mL）。</li>
	<li>根据胃内给药所需的剂量制备浓缩汤剂的21mL等分试样1周，并将等分试样储存在-20°C的冰箱中。</li>
</ol>6.使用强饲装置对小鼠进行管饲
<ol><li>将鼠标放在鼠标笼的表面或其他较粗糙的表面上。</li>
	<li>用右手的拇指和食指抓住鼠标的尾巴。然后，用左手的拇指、食指和中指抓住鼠标的脖子。</li>
	<li>用左手的另外两个手指握住鼠标尾巴，使鼠标的头部、颈部和身体成一条直线。 注意： 确保在整个抓取过程中保持鼠标放松。如果鼠标一直挣扎着移动，则应执行抓取过程，直到鼠标放松。</li>
	<li>右手握住管饲装置，为体重为20g的小鼠（100μL汤剂配方/ 10g体重的小鼠）加载200μL汤剂配方。</li>
	<li>将管饲针从角落插入口腔，并将舌头推到小鼠的上颚上。</li>
	<li>将鼠体保持在同一方向，并沿着食道轻轻地将管饲针推向胃部，直到管饲针留在外面约 10 毫米。 注意：当管饲针进入食道时，有一种空心的感觉。插入小鼠的管饲针的长度应测量到胸骨顶部。管饲针的总长度为60mm（图3），因此，在将汤剂注射到小鼠胃中之前，应将约50mm的管饲针插入小鼠体内。</li>
	<li>将汤剂配方注射到小鼠的胃中。遇到阻力或液体从嘴角流出意味着管饲针错位，必须避免。此外，如果汤剂配方较浓，则必须减慢注射速度。</li>
	<li>松开鼠标并观察它们 10 分钟。如果没有观察到呼吸异常，则管饲成功，并将小鼠放回笼中（图4）。</li>
</ol>7.疗程
<ol><li>为探究益奇节度汤的治疗效果，设置对照组和益奇节度度汤组。对于对照组中的小鼠，给予生理盐水，每天一次，持续28天。对于益奇洁度汤组的小鼠，每天施用益汤28天。</li>
</ol>8.不良反应
<ol><li>仔细观察小鼠被放回笼子后的状态，包括体重减轻和由于食物无法进入胃部而导致的精神状态下降。</li>
	<li>由于食物引起的气管阻塞，小鼠会出现呼吸困难和呼吸急促，并会咳嗽以试图清除气管。如果在管饲过程中观察到任何这些症状，请立即停止管饲并对小鼠实施安乐死。</li>
</ol>

骨肉瘤小鼠的草药煎剂制剂和灌胃标准化

<ol>
	<li>Gao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discussion+on+advantages+and+improvement+of+traditional+Chinese+medicine+decoction.">Discussion on advantages and improvement of traditional Chinese medicine decoction.</a> Chinese Community Physicians (Comprehensive Edition). (21), 12 (2005).</li><li>Chinese Pharmacopoeia Commission. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pharmacopoeia of the People's Republic of China. , (2015).</li><li>An, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpretation+on+specification+of+traditional+Chinese+medicine+decoction.">Interpretation on specification of traditional Chinese medicine decoction.</a> Herald Med. 42 (11), 1648-1652 (2023).</li><li>Chen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+strategies+in+standard+decoction+of+medicinal+slices.">Research strategies in standard decoction of medicinal slices.</a> China J Chinese Materia Medica. 41 (8), 1367-1375 (2016).</li><li>Luo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+soaking+traditional+Chinese+medicine+before+decoction+on+active+components.">Effect of soaking traditional Chinese medicine before decoction on active components.</a> Shanghai Yiyao. 40 (9), 63-64 (2019).</li><li>Liu, F., Chen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+standardization+of+decocting+methods+of+modern+decoction.">On the standardization of decocting methods of modern decoction.</a> Global Trad Chinese Med. 7 (5), 377-378 (2014).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+decocting+times+and+instrument+on+the+quality+of+decoction.">Influence of decocting times and instrument on the quality of decoction.</a> J Trad Chinese Med. 50 (6), 550-552 (2009).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbal+decoctosome+is+a+novel+form+of+medicine.">Herbal decoctosome is a novel form of medicine.</a> Sci China. Life Sci. 62 (3), 333-348 (2019).</li><li>Lan, Y., Hu, Y., Huang, W., Tang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflections+on+PK+-+PD+model+based+on+animal+experiments+for+study+of+Chinese+medicine.">Reflections on PK - PD model based on animal experiments for study of Chinese medicine.</a> Lishizen Med Materia Medica Res. 34 (2), 395-397 (2023).</li><li>Li, Y., Bai, M., Song, Y., Guo, H., Miao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+and+reflection+on+standard+decoction+of+Chinese+materia+medica.">Research and reflection on standard decoction of Chinese materia medica.</a> Chinese Trad Herbal Drugs. 49 (17), 3977-3980 (2018).</li><li>. Estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers Available from: <a target="_blank" href="https://www.fda.gov/regulatory-information/search-fda-guidance-documents/estimating-maximum-safe-starting-dose-initial-clinical-trials-therapeutics-adult-healthy-volunteers">https://www.fda.gov/regulatory-information/search-fda-guidance-documents/estimating-maximum-safe-starting-dose-initial-clinical-trials-therapeutics-adult-healthy-volunteers</a> (2005)</li><li>Chang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratibial+osteosarcoma+cell+injection+to+generate+orthotopic+osteosarcoma+and+lung+metastasis+mouse+models.">Intratibial osteosarcoma cell injection to generate orthotopic osteosarcoma and lung metastasis mouse models.</a> J Vis Exp. (176), 63072 (2021).</li><li>He, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+animal+science.">Laboratory animal science.</a> , (2006).</li><li>Patten, A. R., Fontaine, C. J., Christie, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+different+animal+models+of+fetal+alcohol+spectrum+disorders+and+their+use+in+studying+complex+behaviors.">A comparison of the different animal models of fetal alcohol spectrum disorders and their use in studying complex behaviors.</a> Fronti Pedia. 2, 93 (2014).</li><li>Jones, C. P., Boyd, K. L., Wallace, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+mice+undergoing+serial+oral+gavage+while+awake+or+anesthetized.">Evaluation of mice undergoing serial oral gavage while awake or anesthetized.</a> J Am Assoc Lab Animal Sci. 55 (6), 805-810 (2016).</li><li>Recena Aydos, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonalcoholic+fatty+liver+disease+induced+by+high-fat+diet+in+C57bl/6+models.">Nonalcoholic fatty liver disease induced by high-fat diet in C57bl/6 models.</a> Nutrients. 11 (12), 3067 (2019).</li></ol>

作者声明他们没有什么可披露的。

处方药材在中药中的临床疗效很大程度上与给药剂量和剂型有关;因此，在临床实践中，建立标准化的煎剂策略和熟练的给药过程以及提高实验结果的可靠性是必要的。
本研究首先为中药临床实践中药材标准化汤剂提供了详细的策略，便于引导人们科学用药，也切实提高了中药的汤剂质量和临床疗效;因此，一旦实现标准化，将提高草药疗效在临床实践中的准确性和一致性。</p...

汤剂是中药和药液中最常用的剂型1,2。中药的汤剂配方是将草药浸泡后在水中煎煮，然后过滤以丢弃渣滓。
由于汤剂配方可以根据临床需要进行个体化，特别是符合中医循证治疗的特点，这种经口给药的剂型在临床实践中已有数千年的应用，具有特定且不可替代的优势3,4.汤剂配方的好坏与临床疗效直接相关。因此，必须根据标准化的汤剂方法原理制备汤剂，以确保有效成分得到最大程度的提取和保护3。同时，即饮用药是临床实践中使用的生中草药的主要形式;处方即饮药的质量标准和生产方法必须符合《中国药典》第二版的规定。此外，处方的即食药必须在冷饮水中浸泡至少30分钟，然后才能在特殊的药锅中加热5。研究表明，大部分有效成分可通过煎煮两次来提取，《医疗机构中药煎剂室管理操作规范》也规定煎煮两次4,6,7。如果开了特殊的中草药，例如质地坚硬或滋补品的中草药，建议延长煎煮时间。与汤剂直接接触的汤器皿和储存容器，从浸泡处方即食药物的容器开始，应具有化学稳定性并有盖，例如砂锅菜、陶瓷、玻璃和不锈钢。但是，不应使用铝和普通塑料制品，而应禁止使用铁和其他腐蚀性器皿，以避免可能的化学反应，这会降低治疗效果，甚至产生有害影响4,6,7。
由于进行临床实验的局限性，携带人类疾病的小动物，如小鼠，经常被用于医学研究，以探索不同干预措施的治疗效果和综合机制，包括中药的汤剂配方8,9;然而，与人类不同的是，汤剂配方应每天配制，混合在一起，并在临床实践中每天新鲜给药2次。动物无法自愿按时按规定剂量服药，目前与中药相关的动物实验大多是以管饲法口服的汤剂给药。用于体内动物实验的汤剂配方应提前制备，等分，在-20°C下储存，并在使用前解冻3,4,10。此外，不合格的动物固定和管饲针操作会增加不必要的伤害和负面情绪的风险。因此，作为经口给药的重要方法，熟练的管饲技术对于避免可能的食管损伤和药物溢出尤为重要，从而保证每只模式动物接受等量的处方草药，以达到准确的实验结果。在这里，我们为中药的汤剂配方提供了一个灌胃方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>143B cell line</td>
			<td>ATCC</td>
			<td>CRL-8303</td>
			<td>osteosarcoma cell line</td>
		</tr>
		<tr>
			<td>Anesthesia machine</td>
			<td>Shenzhen RWD Life Technology Co.,Ltd</td>
			<td>R500IP</td>
			<td>The Equipment of Anesthesia mice</td>
		</tr>
		<tr>
			<td>Automatic cell counter</td>
			<td>Shanghai Simo Biological Technology Co., Ltd</td>
			<td>IC1000</td>
			<td>Counting cells</td>
		</tr>
		<tr>
			<td>BALB/c athymic mice</td>
			<td>Shanghai SLAC Laboratory Animal Co, Ltd.</td>
			<td>Male</td>
			<td>Animal</td>
		</tr>
		<tr>
			<td>Barbed skullcap herb</td>
			<td>Shanghai Yanghetang Traditional Chinese Medicine Tablet Co., Ltd.</td>
			<td>20230606</td>
			<td>Decoction-ready medicines&#160;</td>
		</tr>
		<tr>
			<td>Basement Membrane Matrix</td>
			<td>Shanghai Uning BioscienceTechnology Co., Ltd</td>
			<td>356234, BD, Matrigel</td>
			<td>re-suspende cells</td>
		</tr>
		<tr>
			<td>Centrifuge tube (15 mL)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>430790, Corning</td>
			<td>Centrifuge the cells</td>
		</tr>
		<tr>
			<td>Elbow gavage needle (12-gauge/ 55 mm)</td>
			<td>RWD Life Science Co., Ltd.</td>
			<td>C21014-12</td>
			<td>Component of gastric perfusion device</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Haishi Hainuo Group Co., Ltd.</td>
			<td>LC45</td>
			<td>Decoction filter</td>
		</tr>
		<tr>
			<td>Ginseng</td>
			<td>Shanghai Wanshicheng Pharmaceutical Co., Ltd.</td>
			<td>20220704-2</td>
			<td>Decoction-ready medicines&#160;</td>
		</tr>
		<tr>
			<td>Hedyotis diffusa</td>
			<td>Shanghai Yanghetang Traditional Chinese Medicine Tablet Co., Ltd.</td>
			<td>2023071107</td>
			<td>Decoction-ready medicines&#160;</td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>Shenzhen RWD Life Technology Co., Ltd</td>
			<td>VETEASY</td>
			<td>Anesthesia mice</td>
		</tr>
		<tr>
			<td>Micro-volume syringe</td>
			<td>Shanghai high pigeon industry and trade Co., Ltd</td>
			<td>0-50 &#956;L</td>
			<td>Inject precise cells into the tibia</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Beyotime Biotechnology</td>
			<td>ST447</td>
			<td>wash the human osteosarcoma cells</td>
		</tr>
		<tr>
			<td>Rubber bands</td>
			<td>Shanghai Hengfei Biotechnology Co., Ltd.</td>
			<td>XPJ</td>
			<td>Fixing gauze on the measuring cup</td>
		</tr>
		<tr>
			<td>Syringe (1 mL )</td>
			<td>Shanghai Mishawa Medical Industry Co., Ltd.</td>
			<td>SBM0040</td>
			<td>Component of gastric perfusion device</td>
		</tr>
		<tr>
			<td>Trypsin (0.25%)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>25200056, Gibco</td>
			<td>trypsin treatment of cells</td>
		</tr>
	</tbody>
</table>

gavage strategy for decoction formula of traditional chinese medicine in osteosarcoma model mice

汤剂是中医中最常用的剂型，经口给药在临床实践中应用了数千年，其特点是见效快、易吸收、根据患者具体症候进行个体化治疗。汤剂配方的好坏直接关系到中药的临床疗效;因此，汤剂配方的标准化过程对于避免主观因素造成的汤剂质量差异具有重要意义。同时，由于进行临床实验的局限性，携带人类疾病的小动物，如小鼠，经常被用于医学研究，以探索不同干预措施的治疗效果和综合机制，包括中药的汤剂配方。因此，作为一种重要的经口给药方法，熟练的管饲技术对于避免潜在的食管损伤和药物溢出尤为重要，这将确保对每只模型动物施用等量的药物，从而获得准确的实验结果。此外，标准化的汤剂配方制备方法和熟练的管饲策略对于保护动物福利和尽量减少使用动物数量是必要的。在此，我们以益奇节度汤剂在骨肉瘤小鼠模型中报告了煎剂配方和管饲技术的详细标准化过程。通过肿瘤体积评估疗效。该方案将最大限度地保护动物，提高研究数据的可靠性，从而为未来研究中药 汤剂体内的治疗效果和分子机制提供有效的策略。

Decoction formula is the most commonly used dosage form of the traditional Chinese medicine and the liquid medicinal drugs1,2. The decoction formula of traditional Chinese medicine is processed by decocting the herbs in water after being soaked, followed by filtering to discard the dregs.
Because the decoction formula can be individualized based on clinical necessities, especially in line with the characteristics of evidence-based treatment in traditional Chinese medicine, this dosage form via trans-oral administration has been applied in clinical practice for thousands of years with specific and irreplaceable advantages3,4. The quality of the decoction formula is directly related to the clinical efficacy.&#160;Therefore, the decoction formula must be prepared based on the principles for the standardized decoction methods to ensure the active ingredients are maximally extracted and protected3. Meanwhile, decoction-ready medicines are the main form of raw Chinese herbs used in clinical practice; the quality standards and production methods of prescribed decoction-ready medicines must comply with the provisions of the Chinese Pharmacopoeia2. Moreover, the prescribed decoction-ready medicines must be soaked in cold drinking water for at least 30 min before being decocted in a special pharmaceutical pot by heating5. Studies have shown that most of the active ingredients can be extracted by decocting for two times, and the&#160;Code of Practice for the Administration of Decoction Rooms of Traditional Chinese Medicines in Medical Institutions also prescribes decoction to be done twice4,6,7. If special Chinese herbs, such as one with hard texture or tonics, are prescribed, the decoction time is recommended to be extended. The decoction utensils and storage containers that directly come in contact with the decoction formula, starting from the container in which the prescribed decoction-ready medicines are soaked, should be chemically stable and have lids, such as casseroles, ceramics, glass and stainless steel. However, aluminum and ordinary plastic products should not be used, while iron and other corrosive utensils should be prohibited to avoid possible chemical reactions, which will reduce therapeutic efficacy and even produce harmful effects4,6,7.
Due to the limitations of performing clinical experiments, small animals bearing the human diseases, such as mice, are often used in medical research to explore the therapeutic efficacy and comprehensive mechanisms of different interventions including the decoction formula of traditional Chinese medicine8,9; however, unlike humans, the decoction formulas should be prepared every day, mixed together, and freshly administered 2x a day in clinical practice. Animals are unable to voluntarily take their medications on time with the required dosage, and most of the animal experiments related to traditional Chinese medicine are gavage-based oral administration of the decoction formulas at present. The decoction formulas used for in vivo animal experiments should be prepared in advance, aliquoted, stored at -20 &#176;C and thawed just before use3,4,10. Moreover, substandard animal immobilization and gavage needle manipulation increase the risk of unnecessary injuries and negative emotions. Therefore, as an important method of trans-oral drug delivery, the skilled gavage technique is particularly important to avoid possible esophageal injury and drug spillage, thus ensuring that each model animal receives an equal amount of prescribed herbal medicine to achieve accurate experimental results. Here, we provide a gavage protocol for the decoction formula of traditional Chinese medicine.

作者聚焦：用于增强动物研究的标准化草药汤剂方案

中药中药汤剂在骨肉瘤模型小鼠中的强饲策略

以胫骨内异种移植骨肉瘤小鼠模型为研究对象，将143B骨肉瘤细胞注射到无胸腺小鼠胫骨中，测定了益奇节度汤对骨肉瘤 体内 生长的抑制作用。从143B注射的第二天开始，以强饲法给益祺节度汤组小鼠施用益祁节度汤28 d。对照组小鼠给予生理盐水。所有小鼠在28天后被安乐死。结果显示，与对照组相比，益祁节度汤组的肿瘤体积明显减少（图5）。
<p class="jove_content b...

该方案以骨肉瘤小鼠模型中的益奇节度汤提供了详细的汤剂配方和管饲技术标准化流程。它描述了动物保护，提高了研究数据的可靠性，为研究汤剂 在体内的治疗效果和分子机制提供了有效的策略。

Decoction formula is the most commonly used dosage form in traditional Chinese medicine and applied in clinical practice for thousands of years by ...

本研究为循序渐进的预防准备提供了详细和标准化的方案，为有需要的人提供了参考。我们的研究确保每只动物的剂量相等，以产生准确的结果。我们的研究旨在最大限度地保护动物，并提高汤剂制剂研究结果的可靠性。

gavage-strategy-for-decoction-formula-traditional-chinese-medicine

Research

JoVE Journal

Medicine

Gavage Strategy for Decoction Formula of Traditional Chinese Medicine in Osteosarcoma Model Mice

419 次观看.  Shanghai University of Traditional Chinese Medicine. 本研究为循序渐进的预防准备提供了详细和标准化的方案，为有需要的人提供了参考。我们的研究确保每只动物的剂量相等，以产生准确的结果。我们的研究旨在最大限度地保护动物，并提高汤剂制剂研究结果的可靠性。

视频: 作者聚焦：用于增强动物研究的标准化草药汤剂方案

Decoction formula is the most commonly used dosage form in traditional Chinese medicine and applied in clinical practice for thousands of years by trans-oral administration, which is characterized by quick effect, easy absorption, and individualized treatment based on the specific syndromes of patients. The quality of the decoction formula is directly responsible for the clinical efficacy of traditional Chinese medicine; therefore, the standardization process of the decoction formula is important to avoid differences in decoction quality caused by subjective factors. Meanwhile, due to the limitations of performing clinical experiments, small animals bearing human diseases, such as mice, are often used in medical research to explore the therapeutic efficacy and comprehensive mechanisms of different interventions, including the decoction formula for traditional Chinese medicine.&#160;Consequently, as an important trans-oral administration method, the skilled gavage technique is particularly important to avoid potential esophagus damage and drug spillage, which will ensure an equal amount of medicine being administered to each model animal, leading to accurate experimental results. Furthermore, the standardized method of decoction formula preparation and skilled gavage strategy are necessary to protect animal welfare and minimize the number of animals used. Here, we reported a detailed standardization process of the decoction formula and gavage technique with Yiqi Jiedu decoction in osteosarcoma mouse model as an example. The efficacy was evaluated by the tumor volume. This protocol will maximize animal protection and improve the reliability of research data, therefore providing effective strategies for future investigating therapeutic efficacy and molecular mechanisms of decoction formula for traditional Chinese medicine in vivo.

The protocol provides a detailed standardization process of decoction formula and gavage technique with Yiqi Jiedu decoction in the osteosarcoma mouse model as an example. It describes animal protection and improves reliability of research data, providing effective strategies for investigating therapeutic efficacy and molecular mechanisms of decoction formulas in vivo.