本研究得到了国家自然科学基金（81973569、82274207、82104533）、成都中医药大学星林学者研究推进项目（XKTD2022013）和宁夏市重点研发计划（2023BEG02012）的支持。

红景天 采集自中国四川省甘孜藏族自治州甘孜县卓达雪山（北纬31.44570°，东经99.96086°，海拔4892米）。这些植物由成都中医药大学民族医学院的张毅教授鉴定为真品。
1. 红景天的收集
<ol><li>拍摄 红景天的栖息地地图。</li>
	<li>拍摄红景天的整个植物、叶子、花萼和根茎。</li>
	<li>用铁锹清除红 景天 1 m 范围内的杂草和碎石，以确保随后的顺利开采。</li>
	<li>用锄头挖土，直到看到整个根际并收集主根。 注：用于药用部位的 红景天 的根和根茎应在秋季花茎枯萎时采集。</li>
</ol>2.特征识别
<ol><li>用肉眼观察 红景天 的外观特征：圆柱形和短主根和根茎，棕色表面，带有粉红色图案的膜黄色表皮以及橙红色或勃艮第切片。</li>
	<li>通过气味识别它：靠近鼻子时会散发出芬芳的气味。</li>
	<li>按味辨：取一小块根放入口中，先啜饮后咀嚼，略带苦味，后甜。</li>
</ol>3.药粉中淀粉颗粒的显微鉴定
<ol><li>用刷子去除 红景天 表面的土壤，将其放入40°C的烤箱中，每24小时翻动一次草药。 注意：药材的易碎性被认为是干燥水分的标准。</li>
	<li>用粉剂机将干燥的药材粉化，并用药用的3号筛子过滤药粉（见 材料表）。</li>
	<li>取一个干净的载玻片（见 材料表），用解剖针挖出粉末（见 材料表），并将其均匀地放置在载玻片的三分之一处，在2毫米以内。</li>
	<li>使用玻璃滴管（见 材料表）向粉末中加入一滴去离子水。使用镊子（见 材料表）握住盖玻片的一端（见 材料表），快速接触液面并覆盖粉末。 注意： 使用解剖针轻轻混合水和药粉，以确保样品混合均匀。载玻片、粉末和盖玻片之间不应有气泡。</li>
	<li>打开显微镜（参见 材料表）并将步骤3.4中的载玻片放在平台上以固定它。调整光源和粗焦螺旋以查看粉末。调整细小的旁焦点螺旋，直到清楚地看到组织。切换到40倍物镜并观察淀粉颗粒。 注意：对呈现为单个或多个颗粒的谷物进行淀粉，脐带点呈人字形或裂缝状。</li>
</ol>4.药粉中导管、软木细胞、纤维、木实质细胞、色素团的显微鉴定
<ol><li>取一张干净的载玻片（见 材料表），用解剖针挖出粉末（见 材料表），并将其放置在载玻片的三分之一处。</li>
	<li>使用玻璃滴管（见 材料表）向粉末中加入一滴水合氯醛（见 材料表）。用镊子取载玻片（见 材料表），在酒精灯中加热三次，每次加热1秒。 注意： 加热过程中应避免气泡。液体保持不流动，表明渗透已完成。</li>
	<li>使用玻璃滴管加入一滴甘油（见 材料表）。用镊子握住盖玻片的一端，快速接触液位。</li>
	<li>打开显微镜并将载玻片放在平台上以固定它。调整光源和粗焦螺旋以查看粉末。调整细小的旁焦点螺旋，直到清楚地看到组织。通过切换到 40 倍物镜观察导管、软木细胞、纤维、木实质细胞和颜料块。 注：软木细胞多边形或长多边形，螺旋状血管具有明显的螺旋结构，木质部薄壁组织含有草酸钙砂晶，红色或棕红色色素块。</li>
</ol>5. 红景天薄层色谱（TLC）样品的制备及其对照品
<ol><li>将称重纸放在天平上（见 材料表）并称取 3 克 红景天粉末。</li>
	<li>将粉末放入 100 mL 锥形瓶中（参见 材料表），并用大腹移液管加入 25 mL 甲醇（参见 材料表）。将锥形瓶放入超声波仪器中。将功率设置为 250 W，频率设置为 40 kHz，时间设置为 30 分钟（参见 材料表），然后打开仪器。 注：超声仪器的目的是确保 红景天 粉末完全溶解，而不影响后续薄层色谱实验的结果。</li>
	<li>取出锥形瓶，用流水冲洗外瓶至室温 （RT）。</li>
	<li>用 1 mL 注射器吸出步骤 5.3 中制备的 800 μL 液体。用0.22μm微孔滤膜过滤（参见 材料表），并在色谱样品瓶中收集400μL 红景天 中游样品溶液。</li>
	<li>称取并分别加入 2 mg 水景苷、酪醇和没食子酸（参见 材料表）到 3 个单独的 100 mL 锥形瓶中。用大肚移液管向每个锥形瓶中加入 25 mL 甲醇。</li>
	<li>将锥形瓶放入超声波仪器中，将功率设置为250 W，频率设置为40 kHz，时间设置为30分钟，然后重复步骤5.3（见 材料表）。</li>
	<li>用 1 mL 注射器吸出步骤 5.6 中制备的 800 μL 液体。用0.22μm微孔滤膜过滤（见 材料表），并在相应的色谱样品瓶中收集400μL中游水景苷，酪醇和没食子酸标准溶液。</li>
</ol>6. TLC鉴定
<ol><li>移液器三氯甲烷 （5 mL）、乙酸乙酯 （4 mL）、甲醇 （2 mL） 和甲酸 （0.5 mL）（参见 材料表）。加入双罐层析筒的一侧（见 材料表），摇匀并混合。盖住上气缸盖。</li>
	<li>将没食子酸，水景苷，酪醇标准溶液和 红景天 溶液放在样品架上的位置A1-A4。</li>
	<li>将 5 cm x 10 cm 硅胶片（参见 材料表）放在采样台上。启动自动取样机（见 物料表），打开气控阀。</li>
	<li>打开visionCATS软件（参见材料表）。单击“新建”>“新建文件夹（名为红景天样本测试）”>“确定”。单击“新方法”（名称 Rhodiola crenulata 样品测试）> OK > ATS 4。</li>
	<li>单击 “完成步骤定义”（Finish Step Definition）。单击 “跟踪分配 ”以编辑描述（没食子酸、水景苷、酪醇和 红景天 样品）。</li>
	<li>单击 HPTLC 步骤。设置薄层参数 （5 cm x 10 cm）。选择 应用类型 （波段），设置参数（表1），单击 “确定 ”按钮。</li>
	<li>开放式 灌装/冲洗质量。选中 仅填充编程音量。将样品瓶底部液位 （mm） 设置为 0.5，然后单击 OK 按钮。</li>
	<li>单击 “执行方法”。</li>
	<li>单击 “跟踪分配 ”按钮，选中 “居中”，然后设置参数（表 2）。</li>
	<li>单击 “继续 ”按钮进行自动采样。</li>
	<li>关闭自动取样机和空气阀。从自动取样机上取下硅胶片。</li>
	<li>在步骤6.1中将硅胶片放入双罐层析筒的另一侧，盖上上筒盖，预饱和20分钟。</li>
	<li>用镊子轻轻夹住薄层板的上端，迅速将硅胶片放入显影剂中，盖上上气缸盖。 注意： 当展开的前边缘距离薄层板上端 0.5-1.0 厘米时，取出硅胶片。</li>
	<li>有机溶剂蒸发后，在室温下将显色溶液喷洒到硅胶片表面，以获得显色效果。 注：显色溶液是含有 2% FeCl3 和 1% K3[Fe（CN）6] 的水溶液。</li>
</ol>

红景天月状物的显微表征和基于 TLC 的鉴定

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作者没有什么可透露的。

世界上有90多种红景天植物，其中60%以上的物种分布在我国，常见的有红景天、红景天、红景天和红景天17。《中国药典》（2020年）第一部分记载的红景天是一种生长在高海拔地区的传统藏药。红景天的市场需求每年都在增加，因此确保正确使用来源是确保安全使用的关键。特别是，从现场拣选到简单快速的实验室常规鉴定的标准化不容忽视。?...

草药在中国有着悠久的历史和丰富的应用经验，是神农草药经典1中的第一个系统记录。青蒿素应用于疟疾的发现，推动了草药的发展进入一个新的阶段1。利用现代科学技术揭示草药的确切机理，提高了草药的利用率和需求，为其开辟了新的国际市场2,3,4。然而，这导致了一系列负面影响。非专业人士对草药的特性认识模糊，这使得草药的使用面临巨大的安全风险5。
红景天是红景天属植物之一，主要分布于西藏、云南西北部和四川西部（图1）6,7。红景天含有黑景天、酪醇、没食子酸等化合物，具有“活气促血、清脉、镇静哮喘”等功能，用于治疗缺氧相关疾病8,9,10,11。野外调查表明，在海拔4,000-5,600 m的高山距骨带、沟壑斜坡和岩石缝隙中可以找到红景天。它的生长环境寒冷，阳光充足，辐射强烈，属于高寒草甸生态系统。红景天根据生长地形可分布在层状和点状种群中，通过异花授粉进行基因流动。
红景天属的花粉流产、非法挖掘和生态环境的退化使红景天成为濒危物种6,12。鉴于红景天的高药用价值，预计假冒产品将流入市场。本文介绍了红景天的栖息地和一些方便的实验室鉴定方法。首先，我们观察了红景天的生长环境及其药用特性。其次，通过显微镜观察药粉的微观结构;最后一步是关键点。根据红景天在某种物质中的不同吸附或溶解特性，对红景天的代表性成分进行分离和鉴定。基于DNA的药用植物鉴定或代谢组学分析方法复杂且昂贵13.这些基本、方便、经济的方法可以快速识别红景天。

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			<td>0.22 &#956;m millipore filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
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			<td>Automatic sampling machine</td>
			<td>CAMAG</td>
			<td>ATS 4</td>
			<td></td>
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		<tr>
			<td>Chloral hydrate</td>
			<td>Fuzhou Brunei Technology Co., Ltd</td>
			<td>ST1002</td>
			<td></td>
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		<tr>
			<td>Chromatographic&#160;sample&#160;bottles</td>
			<td>Zhejiang ALWSCI Technology Co., Ltd</td>
			<td>C0000008</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical flask</td>
			<td>Sichuan Shubo Co., Ltd</td>
			<td>1121</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Citotest Labware Manufacturing Co., Ltd</td>
			<td>10211818c</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting needle</td>
			<td>Shanghai Bingyu Fluid Technology Co., Ltd</td>
			<td>BY-5026</td>
			<td></td>
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		<tr>
			<td>Electronic balance</td>
			<td>SHIMADZU</td>
			<td>ATX124</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>2022120901</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>2021110801</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallic acid</td>
			<td>Chengdu Herbpurify Co., Ltd</td>
			<td>M-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10010618</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed&#160; crusher</td>
			<td>Beijing Zhongxingweiye Instrument Co., Ltd</td>
			<td>FW-100</td>
			<td></td>
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		<tr>
			<td>Methanol</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>20230108</td>
			<td></td>
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		<tr>
			<td>Microscope</td>
			<td>Chongqing Oprec Nistrument Co.,&#160; Ltd</td>
			<td>B203</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Citotest Labware Manufacturing Co., Ltd</td>
			<td>7105P-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Shanghai Yuejin Medical Equipment Co., Ltd</td>
			<td>DHG-8145</td>
			<td></td>
		</tr>
		<tr>
			<td>Pharmacopoeia sieve</td>
			<td>Hangzhou Xingrun sieve factory</td>
			<td>572423281330</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Changde BKMAM Biotechnology Co., Ltd</td>
			<td>120302008</td>
			<td></td>
		</tr>
		<tr>
			<td>Salidroside</td>
			<td>Chengdu Herbpurify Co., Ltd</td>
			<td>H-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Saturate tank&#160;</td>
			<td>Yancheng Liegu Technology Co., Ltd</td>
			<td>10*20 P-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica gel plate</td>
			<td>Yantai Jiangyou Silica Gel Development&#160; Co., Ltd</td>
			<td>HSG20211227</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloromethane</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>20221013-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer&#160;</td>
			<td>Changde BKMAM Biotechnology Co., Ltd</td>
			<td>130302027</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrosol</td>
			<td>Chengdu Herbpurify Co., Ltd</td>
			<td>L-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound equipment</td>
			<td>Ningbo Xinyi Ultrasonic Equipment Co., Ltd</td>
			<td>SB-8200DTS</td>
			<td></td>
		</tr>
		<tr>
			<td>Volumetric pipet</td>
			<td>Changde BKMAM Biotechnology Co., Ltd</td>
			<td>120301006</td>
			<td></td>
		</tr>
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field collection and laboratory routine identification of rhodiola crenulata

药材鉴定是药品安全的前提和保障。大多数科研人员必然会青睐简单、快速、有效、廉价的草药鉴定过程。 红景天 是一种生长在高海拔地区的传统藏药，主要分布于中国西藏、云南和四川地区。 红景天 具有抗炎、抗缺氧、抗氧化等多种生物活性，具有很大的发展潜力。随着市场需求的增加和资源含量的迅速减少，大量混淆的 红景天 产品一直困扰着人们。因此，该协议引入了在现场鉴定 红景天 的标准过程，并结合常规实验室测试。生境、微观特征和薄层色谱法的结合，无疑将快速、高效、经济地鉴定 红景 天，为藏医药的不断发展和药材的质量控制做出贡献。

Herbal medicine has a long history and rich application experience in China, and it was the first systematic recording in Shennong&#39;s herbal classic1. The discovery of artemisinin applied to malaria promoted the development of herbal medicine into a new stage1. The use of modern scientific technology to uncover the exact mechanism of herbal medicine increases the utilization rate and demand for herbal medicine, opening a new international market for it2,3,4. However, this has led to a series of negative effects. Non-professionals have a vague understanding of the characteristics of herbal medicine, which makes the use of herbal medicine face a huge safety risk5.
Rhodiola crenulata, one of the plants of the Rhodiola species, is mainly distributed in Tibet, northwest Yunnan, and western Sichuan of China (Figure 1)6,7. Rhodiola crenulata comprises salidroside, tyrosol, gallic acid, and other compounds for the treatment of hypoxic-related diseases through the function of &#34;invigorating qi and promoting blood circulation, clearing pulse and calming asthma&#34;8,9,10,11. Field investigation shows that Rhodiola crenulata can be found in the alpine talus zones, gully slopes, and rock crevices at an altitude of 4,000-5,600 m. Its growing environment is cold, full of sunshine and intense radiation, and it belongs to the alpine meadow ecosystem. Rhodiola crenulata can be distributed in lamellar and point-like populations according to the growth terrain, and gene flow can be carried out through cross-pollination.
The pollen abortion of the genus Rhodiola, illegality excavation, and degenerated ecological environment make Rhodiola crenulata an endangered species6,12. In view of the high medicinal value of Rhodiola crenulata, counterfeit products are expected to flow into the market. This article presents the habitat of Rhodiola crenulata and some convenient laboratory identification methods. Firstly, we observed the growth environment of Rhodiola crenulata and its medicinal properties. Secondly, the microstructure of medicinal powder was observed by microscope. The last step is the key point. The representative components of Rhodiola crenulata were separated and identified according to the different adsorption or dissolution properties of these components in a certain substance. DNA-based authentication or metabolomics analysis methods of medicinal plants are complicated and expensive13. These basic, convenient, and economical methods can quickly identify Rhodiola crenulata.

作者聚焦：开发 红景天 鉴定方法并研究其资源分布和药理作用

红景天的现场采集和实验室常规鉴定

The scope of our research is centered on conducting a resource survey of ethnic medicine. Specifically, this study aims to develop a rapid and convenient methods for the identification of Rhodiola crenulata. The protocol we offer presents a cost-effective, straightforward, and rapid method for identifying various medicinal herbal materials.Our laboratory will concentrate its future research endeavors on investigation two primary areas, the distribution of research associated with Rhodiola crenulata and the pharmacological impacts of this species.

该实验方案描述了在田间识别和收集 红景天 。 红景天 倾向于生活在高山距骨带、沟壑斜坡和高海拔的岩石缝隙中。 红景天 的生境、全株、花和叶如 图2所示。 红景天 具有红褐色的根茎（图3A）。药粉的代表性图像如 图3B所示。根据上述实验方案，显微结果可列举如下：1）棕黄色或无色软木细胞，外...

Watch this Scientific Journal Video about Field Collection and Laboratory Routine Identification of Rhodiola crenulata at JoVE.com

在这里，我们从栖息地、植物形态、药用特性、微观特征和薄层色谱法中描述了红 景天 的鉴定。

The identification of medicinal materials is the premise and guarantee of drug safety. The majority of scientific researchers are bound to favor the ...

我们的研究范围集中在进行民族医学的资源调查。具体而言，本研究旨在开发一种快速方便的红景天鉴定方法。我们提供的方案提供了一种具有成本效益、直接和快速的方法来识别各种药材。我们的实验室将把未来的研究工作集中在两个主要领域的调查上，即与红景天相关的研究分布和该物种的药理学影响。

field-collection-laboratory-routine-identification-rhodiola

Research

JoVE Journal

Medicine

Field Collection and Laboratory Routine Identification of Rhodiola crenulata

442 次观看.  Chengdu University of Traditional Chinese Medicine. 我们的研究范围集中在进行民族医学的资源调查。具体而言，本研究旨在开发一种快速方便的红景天鉴定方法。我们提供的方案提供了一种具有成本效益、直接和快速的方法来识别各种药材。我们的实验室将把未来的研究工作集中在两个主要领域的调查上，即与红景天相关的研究分布和该物种的药理学影响。

视频: 作者聚焦：开发 红景天 鉴定方法并研究其资源分布和药理作用

The identification of medicinal materials is the premise and guarantee of drug safety. The majority of scientific researchers are bound to favor the simple, fast, effective, and inexpensive identification process of herbals. Rhodiola crenulata is a traditional Tibetan medicine grown at high altitudes, mainly distributed in Tibet, Yunnan, and Sichuan regions of China. Rhodiola crenulate possesses multiple bioactivities, such as anti-inflammatory, anti-hypoxia, and antioxidant properties, and has great potential for development. With the increasing market demand and a rapid decrease in resource content, a large number of confused products of Rhodiola crenulata have been troubling people. Therefore, this protocol introduces a standard process for the identification of Rhodiola crenulata in the field combined with routine laboratory testing. The combination of habitat, microscopic features, and thin-layer chromatography will undoubtedly identify Rhodiola crenulata quickly, efficiently, and economically, contributing to the continuous development of Tibetan medicine and the quality control of medicinal materials.

Here, we describe the identification of Rhodiola Crenulata from habitat, plant morphology, medicinal properties, microscopic features, and thin-layer chromatography.

科研

医学

Macroscopic and Microscopic Identification of Rhodiola crenulata Traits and Constituents

To begin, observe the appearance traits of Rhodiola Crenulata with the unaided eye and record the observations. Inhale near the plant to identify the fragrance smell. Next, place a small piece of the root in the mouth, then sip and chew for taste identification.Using a brush, remove the soil on the surface of the plant. Place the plant in an oven at 40 degrees Celsius and turn it every 24 hours. Then powder the dried medicinal materials using a powder machine and filter it through a medicated number three sieve.Spread the powder evenly on a clean slide using a dissecting needle covering 1/3 of the slide within a two millimeter area. Using a glass dropper, add a drop of deionized water to the powder and quickly place a cover glass using a tweezer. Next, place the slide onto the microscope platform.Adjust the light source and the coarse focus spiral to view the powder. Switch to a 40X objective lens and observe the starch granules. Again, spread the powder evenly on a slide using a dissecting needle, then add a drop of chloral hydrate to the powder.Hold the slide with a tweezer and heat on the alcohol lamp thrice for one second each. Add a drop of glycerin to the powder and quickly place a cover glass onto the drop. After adjusting the microscope settings as demonstrated previously, select a 40X objective lens to visualize the catheter, cork cells and fibers, along with the wood parenchyma cells and pigment block respectively.Microscopic examination of our Crenulata revealed key features with cork cells appearing brownish yellow, or colorless with polygonal shapes. Starch grains were observed as single or multiples with distinctive umbilical points and closely arranged spiral vessels. Additionally, xylem parenchyma cells were visualized as achromatic and oval, containing calcium oxalate sand crystals, and irregularly shaped brown red pigment blocks were identified.

红景天性状和成分的宏观和微观鉴定

Preparing and Analyzing Thin Layer Chromatography Samples of Rhodiola crenulata and Reference Compounds

To begin, weigh three grams of Rhodiola crenulata powder and transfer it into a 100-milliliter conical bottle. Using a large bowl pipette, add 25 milliliters of methanol into the bottle. Place the bottle in the ultrasonic instrument.Set the power to 250 watts, frequency to 40 kilohertz, time to 30 minutes, and start the ultrasonication. Next, rinse the outside of the bottle under running water until it reaches room temperature. Filter the solution through a 0.22-micron microporus membrane.Using a one-milliliter syringe, aspirate 800 microliters of liquid from the bottle and collect 400 microliters of the midstream solution into a chromatographic sample bottle. Measure and add one milligram of salidroside, 0.5 milligrams of tyrosol, and 0.5 milligrams of gallic acid into three separate one-milliliter volumetric flasks. Then, fill each flask to the scale marked with methanol.Sonicate the samples in the ultrasonic instrument using the conditions described previously. Following ultrasonication, aspirate 800 microliters of liquid from the flask. Filter the solution through a 0.22-micrometer microporous membrane and collect 400 microliters of filtrate into the bottle.Add trichloromethane, ethyl acetate, methanol, and formic acid on one side of the double tank chromatography cylinder. Shake the cylinder to mix the contents evenly and cover the upper cylinder head. Arrange the gallic acid, salidroside, tyrosol standard solutions, and our crenulata solution on the sample rack in positions A1 to A4.Place a 5x10-centimeter silicone sheet on the sampling table.Turn on the automatic sampling machine and open the air control valve. Press the Continue button for automatic sampling. After sampling, turn off the machine and air valve.Remove the silicone sheet from the machine. Put the silicone sheet into the other side of the double tank chromatography cylinder. Cover the upper cylinder head and pre-saturate for 20 minutes.Next, use a tweezer to hold the upper end of the thin layer plate and quickly put the silicone sheet into the development agent. Once the organic solvent evaporates, spray the silicone sheet with chromogenic solution. The thin layer chromatographic analysis showed that the R.crenulata sample appeared in spots of the same color, corresponding to the gallic acid, salidroside, and tyrosol standards.

作者聚焦：开发 红景天 鉴定方法并研究其资源分布和药理作用

作者聚焦：开发红景天鉴定方法并研究其资源分布和药理作用