Name: В центре внимания автора: Разработка методов идентификации родиолы кренулата и исследование ее распределения ресурсов и фармакологических эффектов
Uploaded: 2023-10-27T13:20:00
Description: Watch this Scientific Journal Video about Field Collection and Laboratory Routine Identification of Rhodiola crenulata at JoVE.com

Эта работа была поддержана Национальным фондом естественных наук Китая (81973569, 82274207 и 82104533), Проектом содействия научным исследованиям Xinglin Университета ТКМ в Чэнду (XKTD2022013) и Ключевой программой исследований и разработок Нинся (2023BEG02012).

Родиола зубчатая (Rhodiola crenulata) добывается на снежной горе Чжуода, уезд Ганьцзы, Ганьцзы-Тибетский автономный округ, провинция Сычуань, Китай (N 31.44570°, E 99.96086°, 4892 м). Подлинность растений подтверждена профессором И Чжаном из Школы этнической медицины Университета традиционной китайск?...

Микроскопическая характеристика и идентификация родиолы кренулированной на основе ТСХ

<ol>
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В мире насчитывается более 90 видов родиолы, и более 60% всех видов встречаются в Китае, распространены такие распространены, как родиола кренистая, родиола розовая, родиолас ахалиненский и родиола белая17. Родиола кренистая, зафиксированная в первой части...

Фитотерапия имеет долгую историю и богатый опыт применения в Китае, и это была первая систематическая запись в классической траве Шэньнуна1. Открытие артемизинина применительно к малярии способствовало развитию фитотерапии на новой ступени1. Использование современных научных технологий для раскрытия точного механизма фитотерапии повышает коэффициент использования и спрос на фитотерапию, открывая для нее новый международный рынок 2,3,4. Однако это привело к ряду негативных последствий. Непрофессионалы имеют смутное представление о характеристиках фитотерапии, что ставит использование фитотерапии с огромным риском для безопасности5.
Родиола зубчатая, одно из растений вида Родиола, в основном распространена в Тибете, на северо-западе провинции Юньнань и на западе провинции Сычуань в Китае (рис. 1)6,7. Родиола кренулата содержит салидрозид, тирозол, галловую кислоту и другие соединения для лечения заболеваний, связанных с гипоксией, благодаря функции «оживления ци и улучшения кровообращения, очищения пульса и успокоения астмы»8,9,10,11. Полевые исследования показывают, что родиола зубчатая встречается в альпийских осыпных зонах, овражных склонах и расщелинах скал на высоте 4000-5600 м. Среда его произрастания холодная, полная солнечного света и интенсивной радиации, и он принадлежит к экосистеме альпийских лугов. Родиола кренулатная может быть распределена в пластинчатых и точечных популяциях в зависимости от рельефа произрастания, а поток генов может осуществляться путем перекрестного опыления.
Пыльцевой аборт рода Rhodiola, незаконная выемка и деградация экологической среды делают Rhodiola crenulata исчезающим видом 6,12. В связи с высокой лекарственной ценностью родиолы кренистой (Rhodiola crenulata) ожидается поступление на рынок контрафактной продукции. В данной статье представлен ареал обитания родиолы кренулатной и некоторые удобные лабораторные методы идентификации. Во-первых, мы наблюдали за средой произрастания родиолы кренулатной и ее лечебными свойствами. Во-вторых, микроструктуру лекарственного порошка наблюдали под микроскопом. Последний шаг является ключевым моментом. Репрезентативные компоненты родиолы кренулатной были разделены и идентифицированы в соответствии с различными адсорбционными или растворяющими свойствами этих компонентов в определенном веществе. Методы аутентификации или метаболомного анализа лекарственных растений на основе ДНК сложны и дорогостоящи13. Эти простые, удобные и экономичные методы позволяют быстро определить родиолу кренитовидную.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;m millipore filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic sampling machine</td>
			<td>CAMAG</td>
			<td>ATS 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral hydrate</td>
			<td>Fuzhou Brunei Technology Co., Ltd</td>
			<td>ST1002</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>20230108</td>
			<td></td>
		</tr>
		<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>
	</tbody>
</table>

field collection and laboratory routine identification of rhodiola crenulata

Идентификация лекарственного сырья является предпосылкой и гарантией безопасности лекарственного средства. Большинство научных исследователей склоняются к простому, быстрому, эффективному и недорогому процессу идентификации растительных препаратов. Родиола зубчатая - это традиционная тибетская медицина, выращиваемая на больших высотах, в основном распространенная в Тибете, Юньнани и Сычуань в Китае. Родиола зубчатая обладает множеством биологических свойств, таких как противовоспалительные, антигипоксические и антиоксидантные свойства, и имеет большой потенциал для развития. В связи с растущим рыночным спросом и быстрым снижением ресурсосодержания, большое количество запутанных продуктов родиолы кренулатной беспокоит людей. Таким образом, этот протокол вводит стандартный процесс идентификации родиолы кренулаты в полевых условиях в сочетании с рутинным лабораторным тестированием. Сочетание среды обитания, микроскопических характеристик и тонкослойной хроматографии, несомненно, позволит быстро, эффективно и экономично идентифицировать родиолу кренулату , способствуя постоянному развитию тибетской медицины и контролю качества лекарственных материалов.

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.

В центре внимания автора: Разработка методов идентификации родиолы кренулата и исследование ее распределения ресурсов и фармакологических эффектов

Полевой сбор и лабораторная рутинная идентификация родиолы кренулатной

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.

В этом экспериментальном протоколе описывается идентификация и сбор родиолы зубчатой в полевых условиях. Родиола зубчатая , как правило, обитает в альпийских осыпных зонах, на склонах оврагов и в расщелинах скал на больших высотах. Среда обитания, целое растение, цветок и лист...

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

Здесь мы опишем идентификацию родиолы кренулатной по среде обитания, морфологию растений, лекарственные свойства, микроскопические особенности и тонкослойную хроматографию.

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

Предметом нашего исследования является проведение ресурсного обзора по этнической медицине. В частности, данное исследование направлено на разработку быстрого и удобного метода идентификации родиолы кренистой. Предлагаемый нами протокол представляет собой экономичный, простой и быстрый метод идентификации различных лекарственных растительных материалов.Наша лаборатория сосредоточит свои будущие исследовательские усилия на изучении двух основных областей: распространения исследований, связанных с родиолой кренулатной, и фармакологического воздействия этого вида.

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Research

JoVE Journal

Medicine

Field Collection and Laboratory Routine Identification of Rhodiola crenulata

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.

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.

Макроскопическая и микроскопическая идентификация признаков и составляющих Rhodiola crenulata

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.