Field Collection and Laboratory Routine Identification of Rhodiola crenulata

Summary

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

Abstract

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.

Introduction

Herbal medicine has a long history and rich application experience in China, and it was the first systematic recording in Shennong'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 it^2,3,4. 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 "invigorating qi and promoting blood circulation, clearing pulse and calming asthma"^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 species^6,12. 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.

Protocol

Rhodiola crenulata is collected from Zhuoda Snow Mountain, Ganzi County, Ganzi Tibetan Autonomous Prefecture, Sichuan Province, China (N 31.44570°, E 99.96086°, 4892 m). The plants are authenticated as genuine by Professor Yi Zhang at the School of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine.

1. Collection of Rhodiola crenulata

1. Photograph the habitat map of Rhodiola crenulata.
2. Photograph the whole plant, leaves, calyx, and rhizome of Rhodiola crenulata.
3. Use a spade to clear the weeds and broken stones within 1 m of the Rhodiola crenulata to ensure the subsequent smooth mining.
4. Dig up the soil with a hoe until the whole rhizosphere is seen and collect the taproot.
   NOTE:The roots and rhizomes of Rhodiola crenulata used in medicinal parts should be collected in autumn, when the flower stems wither.

2. Characteristic identification

1. Observe the appearance traits of Rhodiola crenulata with the unaided eye: cylindrical and short taproots and rhizomes, brown surface, membranous yellow epidermis with a pink pattern, and orange-red or burgundy slices.
2. Identify it by smell: It gives a fragrant smell when near the nose.
3. Identify it by taste: Take a small piece of root into the mouth, first sip and then chew, taste slightly bitter, then sweet.

3. Microscopic identification of starch granules in medicinal powder

1. Remove the soil on the surface of the Rhodiola crenulata with a brush, put it in the oven at 40 °C, and turn the herbs every 24 h.
   NOTE: The ease of breaking of medicinal materials is considered the standard for drying moisture.
2. Powder the dried medicinal materials using a powder machine and filter the medicinal powder using the medicated No. 3 sieve (see Table of Materials).
3. Take a clean slide (see Table of Materials), dig the powder with a dissecting needle (see Table of Materials), and place it evenly at one-third of the slide within 2 mm.
4. Use a glass dropper (see Table of Materials) to add a drop of deionized water to the powder. Use a tweezer (see Table of Materials) to hold one end of the cover glass (see Table of Materials) to touch the liquid level quickly and cover the powder.
   NOTE: Use an anatomical needle to gently mix water and medicinal powder to ensure uniform sample mixing. There should be no air bubbles between the slide, powder, and cover glass.
5. Open the microscope (see Table of Materials) and place the slide in step 3.4 onto the platform to secure it. Adjust the light source and the coarse focus spiral to see the powder. Adjust the fine parafocal spiral until the tissues are seen clearly. Switch to a 40x objective and observe the starch granules.
   ​NOTE: Starch the grains presenting as single or multiple grains, and the umbilical point appears herringbone or crack-shaped.

4. Microscopic identification of catheters, cork cells, fibers, wood parenchyma cells, and pigment masses in medicinal powder

1. Take a clean slide (see Table of Materials), dig the powder with a dissecting needle (see Table of Materials), and place it at one-third of the slide.
2. Use a glass dropper (see Table of Materials) to add a drop of chloral hydrate (see Table of Materials) to the powder. Take the slide with a tweezer (see Table of Materials) and heat it in the alcohol lamp three times, each time for 1 s.
   NOTE: Bubbles should be avoided during heating. The liquid remains non-flowing, indicating that the penetration is complete.
3. Use a glass dropper to add a drop of glycerin (see Table of Materials). Use a tweezer to hold one end of the cover glass to touch the liquid level quickly.
4. Open the microscope and place the slide onto the platform to secure it. Adjust the light source and the coarse focus spiral to see the powder. Adjust the fine parafocal spiral until the tissues are seen clearly. Observe the catheter, cork cells, fibers, wood parenchyma cells, and pigment block by switching to a 40x objective lens.
   ​NOTE: Polygonal or long polygonal of cork cells, spiral vessel with obvious helical structure, xylem parenchyma containing calcium oxalate sand crystals, and red or brownish-red pigment block.

5. Preparation of thin layer chromatographic (TLC) samples of Rhodiola crenulata and its reference

1. Place the weighing paper on the balance (see Table of Materials) and weigh 3 g powder of Rhodiola crenulata.
2. Take the powder into a 100 mL conical bottle (see Table of Materials), and add 25 mL of methanol with a big belly pipette (see Table of Materials). Put the conical bottle into the ultrasonic instrument. Set the power to 250 W, frequency to 40 kHz, and time to 30 min (see Table of Materials), and switch on the instrument.
   NOTE: The purpose of the ultrasound instrument is to ensure that the powder of Rhodiola crenulata is completely dissolved without affecting the results of subsequent thin-layer chromatographic experiments.
3. Remove the conical bottle and rinse the outer bottle with running water to room temperature (RT).
4. Aspirate 800 µL of liquid prepared in step 5.3 with a 1 mL syringe. Filter with 0.22 µm microporous filter membrane (see Table of Materials) and collect 400 µL of midstream sample solution of Rhodiola crenulata in a chromatographic sample bottle.
5. Weigh and add 2 mg of salidroside, tyrosol, and gallic acid (see Table of Materials) into 3 separate 100 mL conical bottles, respectively. Add 25 mL of methanol with a big belly pipette into every conical bottle.
6. Put the conical bottle into the ultrasonic instrument, set the power to 250 W, frequency to 40 kHz, and time to 30 min, and repeat step 5.3 (see Table of Materials).
7. Aspirate 800 µL of liquid prepared in step 5.6 with a 1 mL syringe. Filter with 0.22 µm microporous filter membrane (see Table of Materials) and collect 400 µL of midstream salidroside, tyrosol, and gallic acid standard solution in corresponding chromatographic sample bottles.

6. TLC identification

1. Pipette trichloromethane (5 mL), ethyl acetate (4 mL), methanol (2 mL), and formic acid (0.5 mL) (see Table of Materials). Add to one side of the double-tank chromatography cylinder (see Table of Materials), shake, and mix evenly. Cover the upper cylinder head.
2. Place the gallic acid, salidroside, tyrosol standard solution, and Rhodiola crenulata solution on the sample rack in positions A1-A4.
3. Place the 5 cm x 10 cm silicone sheet (see Table of Materials) on the sampling table. Start the automatic sampling machine (see Table of Materials), and open the air control valve.
4. Open the visionCATS software (see Table of Materials). Click New > New Folder (named Rhodiola crenulata sample test) > OK. Click New Method (name Rhodiola crenulata sample test) > OK > ATS 4.
5. Click Finish Step Definition. Click Track Assignment to edit the description (gallic acid, salidroside, tyrosol, and Rhodiola crenulata sample).
6. Click HPTLC Steps. Set the thin layer parameters (5 cm x 10 cm). Select Application Type (band), set the parameters (Table 1), and click the OK button.
7. Open Filling/Rinsing Quality. Check Fill only Programmed Volume. Set the vial bottom level (mm) to 0.5, and click the OK button.
8. Click Execute Method.
9. Click the Track Assignment button, check Center, and set the parameters (Table 2).
10. Click the Continue button for automatic sampling.
11. Turn off the automatic sampling machine and air valve. Remove the silicone sheet from the automatic sampling machine.
12. Put the silicone sheet into the other side of the double tank chromatography cylinder in step 6.1, cover the upper cylinder head, and pre-saturate for 20 min.
13. Gently clamp the upper end of the thin layer plate with a tweezer, quickly put the silicone sheet into the development agent, and cover the upper cylinder head.
    NOTE: Take out the silicone sheet when the unfolding front edge is 0.5-1.0 cm away from the upper end of the thin layer plate.
14. After the organic solvent evaporates, spray the chromogenic solution onto the surface of the silicone sheet at room temperature to obtain chromogenic results.
    NOTE: The chromogenic solution is an aqueous solution containing 2% FeCl₃ and 1% K₃[Fe(CN)₆].

Results

This experimental protocol describes identifying and collecting Rhodiola crenulate in the field. Rhodiola crenulate tends to live in the alpine talus zones, gully slopes, and rock crevices at high altitudes. The habitat, whole plant, flower, and leaves of Rhodiola crenulate can be shown in Figure 2. Rhodiola crenulate has a reddish-brown rhizome (Figure 3A). A representative image of the medicinal powder is shown in

Discussion

There are more than 90 species of Rhodiola plants in the world, and more than 60% of all species are found in China, common ones including Rhodiola crenulata, Rhodiola rosea, Rhodiolas achalinensis, and Rhodiola algida¹⁷. Rhodiola crenulata, recorded in the first part of the Chinese Pharmacopoeia (2020), is a traditional Tibetan medicine grown at high altitudes. The market demand for Rhodiola crenulata is increasing yearly, so ensuring the correct use o...

Materials

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