This work was supported by the National Natural Science Foundation of China (81973569, 82274207 and 82104533), the Xinglin Scholar Research Promotion Project of Chengdu University of TCM (XKTD2022013), and the Key Research and Development Program of Ningxia (2023BEG02012).

Rhodiola crenulata is collected from Zhuoda Snow Mountain, Ganzi County, Ganzi Tibetan Autonomous Prefecture, Sichuan Province, China (N 31.44570&#176;, E 99.96086&#176;, 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
<ol>
	<li>Photograph the habitat map of Rhodiola crenulata.</li>
	<li>Photograph the whole plant, leaves, calyx, and rhizome of Rhodiola crenulata.</li>
	<li>Use a spade to clear the weeds and broken stones within 1 m of the Rhodiola crenulata to ensure the subsequent smooth mining.</li>
	<li>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.</li>
</ol>
2. Characteristic identification
<ol>
	<li>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.</li>
	<li>Identify it by smell: It gives a fragrant smell when near the nose.</li>
	<li>Identify it by taste: Take a small piece of root into the mouth, first sip and then chew, taste slightly bitter, then sweet.</li>
</ol>
3. Microscopic identification of starch granules in medicinal powder
<ol>
	<li>Remove the soil on the surface of the Rhodiola crenulata with a brush, put it in the oven at 40 &#176;C, and turn the herbs every 24 h. 
	NOTE: The ease of breaking of medicinal materials is considered the standard for drying moisture.</li>
	<li>Powder the dried medicinal materials using a powder machine and filter the medicinal powder using the medicated No. 3 sieve (see Table of Materials).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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. 
	&#8203;NOTE: Starch the grains presenting as single or multiple grains, and the umbilical point appears herringbone or crack-shaped.</li>
</ol>
4. Microscopic identification of catheters, cork cells, fibers, wood parenchyma cells, and pigment masses in medicinal powder
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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. 
	&#8203;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.</li>
</ol>
5. Preparation of thin layer chromatographic (TLC) samples of Rhodiola crenulata and its reference
<ol>
	<li>Place the weighing paper on the balance (see Table of Materials) and weigh 3 g powder of Rhodiola crenulata.</li>
	<li>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.</li>
	<li>Remove the conical bottle and rinse the outer bottle with running water to room temperature (RT).</li>
	<li>Aspirate 800 &#181;L of liquid prepared in step 5.3 with a 1 mL syringe. Filter with 0.22 &#181;m microporous filter membrane (see Table of Materials) and collect 400 &#181;L of midstream sample solution of Rhodiola crenulata in a chromatographic sample bottle.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Aspirate 800 &#181;L of liquid prepared in step 5.6 with a 1 mL syringe. Filter with 0.22 &#181;m microporous filter membrane (see Table of Materials) and collect 400 &#181;L of midstream salidroside, tyrosol, and gallic acid standard solution in corresponding chromatographic sample bottles.</li>
</ol>
6. TLC identification
<ol>
	<li>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.</li>
	<li>Place the gallic acid, salidroside, tyrosol standard solution, and Rhodiola crenulata solution on the sample rack in positions A1-A4.</li>
	<li>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.</li>
	<li>Open the visionCATS software (see Table of Materials). Click New &#62; New Folder (named Rhodiola crenulata sample test) &#62; OK. Click New Method (name Rhodiola crenulata sample test) &#62; OK &#62; ATS 4.</li>
	<li>Click Finish Step Definition. Click Track Assignment to edit the description (gallic acid, salidroside, tyrosol, and Rhodiola crenulata sample).</li>
	<li>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.</li>
	<li>Open Filling/Rinsing Quality. Check Fill only Programmed Volume. Set the vial bottom level (mm) to 0.5, and click the OK button.</li>
	<li>Click Execute Method.</li>
	<li>Click the Track Assignment button, check Center, and set the parameters (Table 2).</li>
	<li>Click the Continue button for automatic sampling.</li>
	<li>Turn off the automatic sampling machine and air valve. Remove the silicone sheet from the automatic sampling machine.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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].</li>
</ol>

Microscopic Characterization and TLC-Based Identification of Rhodiola crenulate

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The authors have nothing to disclose.

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...

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.

<table><tbody><tr><td>0.22 &amp;mu;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&amp;nbsp;sample&amp;nbsp;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&amp;nbsp; 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.,&amp;nbsp; 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&amp;nbsp;</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&amp;nbsp; 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&amp;nbsp;</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

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.

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 <strong cl...

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

Author Spotlight: Developing Identification Methods for Rhodiola crenulata and Investigating Its Resource Distribution and Pharmacological Effects

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

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 ...

field-collection-laboratory-routine-identification-rhodiola

Research

JoVE Journal

Medicine

577 Views.  Chengdu University of Traditional Chinese Medicine. De reikwijdte van ons onderzoek is gericht op het uitvoeren van een bronnenonderzoek naar etnische geneeskunde. Concreet is deze studie gericht op het ontwikkelen van een snelle en gemakkelijke methode voor de identificatie van Rhodiola crenulata. Het protocol dat we aanbieden biedt een kosteneffectieve, eenvoudige en snelle methode voor het identificeren van verschillende medicinale kruidenmaterialen.Ons laboratorium zal zijn toekomstige onderzoeksinspanningen concentreren op het onde...

Video: Auteur in de schijnwerpers: het ontwikkelen van identificatiemethoden voor Rhodiola crenulata en het onderzoeken van de verdeling van hulpbronnen en farmacologische effecten

Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts

To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral extraction methods and coverslip bioassays were developed utilizing Colletotrichum fioriniae. In blueberry and cranberry, this pathogen is optimally controlled by applying fungicides during the bloom period because of the role flowers play in the initial stages of infection. The protocol detailed here describes how floral extracts (FE) were obtained using water-, chloroform-, and field rainwater-based methods for later use in corresponding glass coverslip bioassays. Each FE served to provide a different set of information: response of C. fioriniae to mobilized floral chemical cues in water (water-based), pathogen response to flower and fruit surface waxes (chloroform-based), and field-based monitoring of collected floral rainwater, moving in vitro observations to an agricultural setting. The FE is broadly described as either water- or chloroform-based, with an appropriate bioassay described to compensate for the inherent differences between these two materials. Rainwater that had run off flowers was collected in unique devices for each crop, alluding to the flexibility and application of this approach for other crop systems. The bioassays are quick, inexpensive, simple, and provide the ability to generate spatiotemporal and site-specific information about the presence of stimulatory floral compounds from various sources. This information will ultimately better inform disease management strategies, as FE decrease the time needed for infection to occur, thus providing insight into changing risks for pathogen infection over the growing season.

Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts

Here, bioassays designed to monitor the development of a fungal pathogen, Colletotrichum fioriniae, in the presence of blueberry or cranberry floral extracts on glass coverslips are described. Water-, chloroform-, and field rainwater- based floral extraction techniques are detailed as well as insight into how this information can be applied.

To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral ...

Environment

High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling in soils, to pathogen competition, plant growth promotion, and cross-kingdom signaling. Furthermore, siderophores are also of commercial interest in bioleaching and bioweathering of metal-bearing minerals and ores. A rapid, cost effective, and robust means of quantitatively assessing siderophore production in complex samples is key to identifying important aspects of the ecological ramifications of siderophore activity, including, novel siderophore producing microbes. The method presented here was developed to assess siderophore activity of in-tact microbiome communities, in environmental samples, such as soil or plant tissues. The samples were homogenized and diluted in a modified M9 medium (without Fe), and enrichment cultures were incubated for 3 days. Siderophore production was assessed in samples at 24, 48, and 72 hours (h) using a novel 96-well microplate CAS (Chrome azurol sulphonate)-Fe agar assay, an adaptation of the traditionally tedious and time-consuming colorimetric method of assessing siderophore activity, performed on individual cultivated microbial isolates. We applied our method to 4 different genotypes/Lines of wheat (Triticum aestivum L.), including Lewjain, Madsen, and PI561725, and PI561727 commonly grown in the inland Pacific Northwest. Siderophore production was clearly impacted by the genotype of wheat, and in the specific types of plant tissues observed. We successfully used our method to rapidly screen for the influence of plant genotype on siderophore production, a key function in terrestrial and aquatic ecosystems. We produced many technical replicates, yielding very reliable statistical differences in soils and within plant tissues. Importantly, the results show the proposed method can be used to rapidly examine siderophore production in complex samples with a high degree of reliability, in a manner that allows communities to be preserved for later work to identify taxa and functional genes.

We present a protocol for rapid screening of environmental samples for siderophore potential contributing to micronutrient bioavailability and turnover in terrestrial systems.

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling ...

Comparative Karyotype Analysis of Lycoris aurea Herb Populations Using Fluorochrome Banding and 45S and 5S rDNA-FISH

To understand karyotype variation in eight populations, detailed karyotypes were meticulously established using chromosomal measurements, fluorescence bands, and rDNA FISH signals. The number of 45S rDNA sites varies from one to five pairs per population, with the most common number per karyotype being four pairs. The 45S rDNA locus is predominantly located in the short arms and terminal regions of chromosomes, while the 5S rDNA locus is found mainly in the short arm and the terminal or proximal regions. Populations HBWF, HNXN, HBBD, and HNZX showed a similar distribution of 45S rDNA sites, as did GXTL, HBFC, and SCLS, indicating a close relationship between populations with similar 45S rDNA site distributions. The karyotypes of all studied populations are symmetrical, comprising stable and metastable centromeres or exclusively stable centromeres. Scatter plots of MCA and CVCL effectively distinguish their karyotypic structures. The analysis includes six quantitative parameters (x, 2n, TCL, MCA, CVCL, CVCI). Additionally, the results indicate that PCoA based on these six parameters is a robust method for determining biological karyotype relationships among the eight populations. The chromosome number in Lycoris populations is x = 6-8. Based on the current study and literature, genomic differentiation of these populations is discussed in terms of genome size, heterochromatin, 45S and 5S rDNA sites, and karyotype asymmetry.

This protocol investigates the heterochromatin distribution in eight populations of Lycoris aurea (L&#8242;Her) Herb, using combined PI and DAPI chromosome staining, fluorescence in situ hybridization (FISH) with 18S-5.8-26S rRNA gene regions (45S rDNA regions), and 5S ribosomal DNA probes.

To understand karyotype variation in eight populations, detailed karyotypes were meticulously established using chromosomal measurements, fluorescence ...

Genetics

HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis

A method for identifying Chinese medicinal materials and their related adulterants was constructed by taking Clematidis Armandii Caulis (Chuanmutong, a universally used traditional Chinese medicine) as an example. Ten batches of genuine Chuanmutong varieties and five batches of related adulterants were analyzed and compared based on the high-performance liquid chromatography (HPLC) fingerprints combined with chemometrics, including cluster analysis (CA), principal component analysis (PCA), and orthogonal partial least-squares discrimination analysis (OPLS-DA). In addition, the content of &#946;-sitosterol was determined. The control chemical fingerprint of Chuanmutong was established, and 12 common peaks were identified. The similarity between the fingerprint of 10 batches of genuine Chuanmutong varieties and the control fingerprint was 0.910-0.989, while the similarity of five batches of adulterants was only 0.133-0.720. Based on the common peaks in the chromatogram, 15 batches of samples were classified into three content levels by PCA, and were aggregated into four categories by CA, achieving a clear distinction between authentic Chuanmutong and adulterants of Chuanmutong. Further, seven differential components that can effectively identify authentic Chuanmutong and adulterants of Chuanmutong were found through OPLS-DA. The &#946;-sitosterol content of 10 batches of genuine Chuanmutong varieties was 97.53-161.56 &#181;g/g, while the &#946;-sitosterol content of the five batches of adulterants varied greatly, among which the &#946;-sitosterol content of Clematis peterae Hand.-Mazz. and Clematis gouriana Roxb. Var. finetii Rehd. et Wils. was significantly lower than that of authentic varieties of Chuanmutong. The HPLC index component content and chemical fingerprint multi-pattern recognition method established in this study provide a new strategy for effectively identifying authentic Chinese medicinal materials and related adulterants.

HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis

Here, we present a protocol to establish high-performance liquid chromatography (HPLC), coupled with chemical fingerprint multi-pattern recognition, which provides a new strategy for effectively identifying the genuine varieties of Clematidis Armandii Caulis and its adulterants.

A method for identifying Chinese medicinal materials and their related adulterants was constructed by taking Clematidis Armandii Caulis (Chuanmutong, a ...

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Chemistry

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.

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.

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

Identifying Chemical Composition of Gynura bicolor by UPLC-TOF Mass Spectrometry

High-resolution Tandem Mass Spectrometry for Studying Chemical Constituents of Gynura bicolor DC

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) has gradually become a leading technology for the identification of TCM ingredients. Gynura bicolor DC. (BFH), a perennial stemless herb used for medicine and food in China has medicinal effects such as clearing heat, moistening the lung, relieving cough, dispersing stasis, and relieving swelling. Polyphenols and flavonoids contain numerous isomers, which hinder the identification of the complex compounds in BFH. This paper presents a systematic protocol for studying chemical constituents of BFH based on solvent extraction and integrated data via UPLC-Q-TOF-MS.
The method described here includes systematic protocols for sample pretreatment, MS calibration, MS acquisition, data processing, and analysis of results. Sample pretreatment includes collection, cleaning, drying, crushing, and extraction. MS calibration consists of multipoint and single-point correction. Data processing includes data importing, method establishment, analysis processing, and result presentation. Representative results of the typical fragmentation pattern of phenolic acids, esters, and glycosides in Gynura bicolor DC. (BFH) are presented in this paper. In addition, organic solvent selection, extraction, data integration, collision energy selection, and method improvement are discussed in detail. This universal protocol can be widely used to identify complex compounds in TCM.

Preparation of Gynura bicolor DC samples for High-Resolution Tandem Mass Spectrometry

We describe a general protocol that integrates high-resolution mass spectrometry analysis and molecular docking in traditional Chinese medicine research.

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). ...

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.

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

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

Biology

Field Identification of Matricaria chamomilla using a Portable qPCR System

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

Field Identification of Matricaria chamomilla using a Portable qPCR System

Presented here is a protocol for field identification of Matricaria chamomilla using a portable qPCR system. This easy-to-perform protocol is ideal as a method to confirm the identity of a botanical species at locations where access to laboratory equipment and expertise is limited, such as farms and warehouses.

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...

Field Collection and Laboratory Routine Identification of Rhodiola crenulata

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