This work was supported by introduces the talented person scientific research start funds subsidization project of Chengdu University of Traditional Chinese Medicine (030040015).

1. Sample preparation
<ol>
	<li>The present study utilized two-year-old A. sinensis with low temperature and long sunshine at an altitude of 2800 m from Min County, Gansu Province, as experimental materials.Three plants were selected from which more than three roots were selected for experimentation. Rinse the roots first with sterilized water and then clean them with an alcohol sprayer or alcohol swabs to prevent contamination that could affect the accuracy of the experimental results. Cut th...

DNA Barcoding for Medicinal Plants: Identification and Phylogenetic Analysis

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Molecular identification technology is easier to learn and master than traditional identification methods. It overcomes the limitations of traditional medicinal herb identification, as it is not affected by the plant&#39;s growth stage and does not rely on subjective judgment or the accumulation of specialized expertise35. Other species are confused with A. sinensis due to its similar morphology and medicinal parts, but with the use of DNA barcoding, it&#160;can be definitively identified...

Medicinal plants have a wide range of pharmacological effects and are important substances for the treatment and prevention of diseases. The market demand for medicinal plants in herbal medicines and pharmaceutical products is huge and still growing. With the increasing market for medicinal plants, the problem of adulteration has hindered the development of medicinal plants. Currently, medicinal plant adulteration suffers from these reasons: 1) the similar morphology of medicinal plants makes it difficult to identify and use them correctly1,2,3,4,5, 2) the increasing demand for medicinal plants has led to insufficient supply in the market6,7, 3) medicinal plants are expensive and have fluctuating prices, and the use of cheap herbs instead of economically valuable materials has led to adulteration and market profiteering8,9. To solve the problems of proper identification and use of medicinal plants, there is a need for a technology that allows non-specialists to identify the source10.
There are limitations to properly identifying and using medicinal plants by appearance and odor alone11. For more accurate identification and quality control, physicochemical methods are employed12. Thin-layer chromatography (TLC), for instance, is a rapid method for identifying medicinal herbs and is included in the Chinese Pharmacopoeia. Nevertheless, it requires reference standards to identify the plants13. High-performance liquid chromatography (HPLC) can perform both qualitative and quantitative tests, making it suitable for medicine quality testing. However, this instrument is expensive and requires specialized knowledge to operate14,15. DNA barcoding technology is a rapid and accurate species identification technology that identifies species by using stable DNA sequences to differentiate between plants with similar morphology. Hebert et al. proposed DNA barcoding technology to identify species, which uses a recognized and relatively short DNA sequence within the genome16, Chen et al. proposed ITS2 as a universal sequence for the molecular identification of medicinal plants and identified more than 6600 plants and found a high identification ability17. The study of medicinal plants based on ITS2 and chloroplast gene fragments has demonstrated the ability to distinguish species at the species level, with applications in the fields of resource protection, market regulation, and international trade17,18,19. The application of DNA barcoding can overcome the limitations of traditional identification methods, which is helpful in ensuring the quality and safety of medicinal plants, preventing resource abuse and confusion20. It has proven beneficial for studying medicinal plants, supporting the growth of the herbal industry in a sustainable manner, and providing a guarantee that the consumer uses the correct medication21,22,23,24.
The paper outlines protocols for how to apply DNA barcoding to identify medicinal plants using A. sinensis as an example. DNA barcoding utilizes one or more standardized short genetic markers in an organism&#39;s DNA to recognize it as belonging to a particular species. Current methods for identifying medicinal plants have certain limitations. Traditional morphological identification relies heavily on experts&#39; extensive experience, which can be influenced by human factors, while chemical identification is prone to issues like adulteration of key compounds. In contrast, DNA barcoding provides a more accurate means of species identification, offering advantages such as speed, high reproducibility, and stability. Furthermore, this technology enables centralized management and sharing of existing species sequence data through the internet and information platforms, significantly improving the efficiency and reliability of species identification11,12. Due to their similar morphology and medicinal parts, A. sinensis is often confused with other species and is frequently misused or intentionally substituted on the market25. Yang et al. identified A. anomala using DNA barcoding to distinguish it from false drugs26. Yuan et al. collected 23 species of Angelica and used DNA barcoding to differentiate between the various species27. By following the procedures described in this protocol, users will be able to authenticate plant-based medicinal materials from pre-processing to ultimately matching the barcoding sequence with the barcode database (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CTAB Rapid Plant Genomic DNA Extraction Kit</td>
			<td>Shanghai Huiling Biotechnology Co.</td>
			<td>NG411M</td>
			<td>Suitable for rapid extraction of high quality genomic DNA from different tissues of a wide range of plants</td>
		</tr>
		<tr>
			<td>DL5000 DNA Marker</td>
			<td>Nanjing vazyme Bio-technology Co.</td>
			<td>MD102-02</td>
			<td>Ready-to-use product, take an appropriate amount of this product directly for electrophoresis when running the gel.</td>
		</tr>
		<tr>
			<td>Electrophoresis</td>
			<td>Beijing Liuyi Biotechnology Co.</td>
			<td>DYY-6C</td>
			<td>Adopt touch screen design, can display set voltage, set current at the same time, parallel output</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>BeijingpsaitongBiotechnologyCo.,Ltd</td>
			<td>E70015-100G</td>
			<td>Nuclear Isolation Buffer formulation reagents.</td>
		</tr>
		<tr>
			<td>Goldview Nucleic Acid Gel Stain(10,000&#215;)</td>
			<td>Yisheng Biotechnology (Shanghai) Co., Ltd</td>
			<td>10201ES03</td>
			<td>When using agarose gel electrophoresis to detect DNA, it binds to DNA and produces a strong fluorescent signal.</td>
		</tr>
		<tr>
			<td>High-Speed Tabletop Centrifuge</td>
			<td>Changsha High-tech Industrial Development Zone Xiangyi Centrifuge Instrument Co.</td>
			<td>H1650</td>
			<td>For fast and efficient separation of samples, this compact and lightweight centrifuge offers reliable safety</td>
		</tr>
		<tr>
			<td>High-Throughput Tissue Grinder</td>
			<td>Shanghai Jingxin Industrial Development Co.</td>
			<td>Tiss-48</td>
			<td>A high-frequency vibration instrument for grinding samples</td>
		</tr>
		<tr>
			<td>http://www.gpgenome.com/</td>
			<td>Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences</td>
			<td>-</td>
			<td>HerbGenomics database includes genome sequences, gene sets, organelle genomes, low coverage genome data and DNA barcode sequences.</td>
		</tr>
		<tr>
			<td>https://www.ncbi.nlm.nih.gov/</td>
			<td>U.S. National Library of Medicine</td>
			<td>-</td>
			<td>The National Center for Biotechnology Information advances science and health by providing access to biomedical and genomic information.</td>
		</tr>
		<tr>
			<td>Kylin-Bell</td>
			<td>Haimen Qilinbel Instrument Manufacturing Co.</td>
			<td>VORTEX-5</td>
			<td>Rapid mixing in the form of a high-speed vortex, mixing speed, uniformity, thoroughness</td>
		</tr>
		<tr>
			<td>LifeTouch</td>
			<td>Hangzhou BORI Technology Co.</td>
			<td>TC-96/G/H(b)B</td>
			<td>Adoption of advanced thermoelectric refrigeration technology and newly created TAS technology to enhance its overall performance</td>
		</tr>
		<tr>
			<td>Multi-functional Gel Image Analysis System</td>
			<td>Southwest Operation Center of Shanghai Tianneng Life Science Co.</td>
			<td>Tanon-Mini Space 2000</td>
			<td>Performs rapid gene amplification experiments with a gradient function for mapping amplification conditions and a gradient temperature range of up to 30&#176;C</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Beijing Solarbio Science&#38;Technology Co.,Ltd.</td>
			<td>S8210-100</td>
			<td>Nuclear Isolation Buffer formulation reagents.</td>
		</tr>
		<tr>
			<td>Nanodrop One</td>
			<td>Genes Ltd.</td>
			<td>ND ONE</td>
			<td>Quantify DNA, RNA and protein samples in seconds with just 1-2 &#181;L of sample</td>
		</tr>
		<tr>
			<td>Polyvinyl pyrrolidone</td>
			<td>Shanghai yuanye Bio-Technology Co., Ltd</td>
			<td>S30268-500g</td>
			<td>Nuclear Isolation Buffer formulation reagents.</td>
		</tr>
		<tr>
			<td>Snowflake Ice Maker</td>
			<td>Shanghai Zhixin Experimental Instrument Technology Co.</td>
			<td>ZX-60X</td>
			<td>Adopting rotary extrusion ice making method, fast ice making speed and high efficiency of ice production</td>
		</tr>
		<tr>
			<td>Stainless Steel Beads for Tissue Homogenizer</td>
			<td>Beyotime Biotechnology.&#160;</td>
			<td>F6623</td>
			<td>Equipment for grinding and mixing of tissue and other samples by vibration</td>
		</tr>
		<tr>
			<td>Tris Acetate-EDTA buffer</td>
			<td>Beyotime Biotech Inc</td>
			<td>ST716</td>
			<td>TAE is a commonly used buffer for DNA electrophoresis, frequently employed in agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Beijing Solarbio Science&#38;Technology Co.,Ltd.</td>
			<td>T8230</td>
			<td>Nuclear Isolation Buffer formulation reagents.</td>
		</tr>
		<tr>
			<td>Water bath Kettle</td>
			<td>Shanghai Senxin Experimental Instrument Co.</td>
			<td>DK-8D</td>
			<td>Precise thermostat and temperature regulation, accurate and reliable temperature control</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Shanghai Eon Chemical Technology Co.</td>
			<td>R054186-100ml&#160;&#160;</td>
			<td>Nuclear Isolation Buffer formulation reagents.</td>
		</tr>
	</tbody>
</table>

application of dna barcoding to identify medicinal plants

Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration obstructs their development. DNA barcoding, a technique for species identification by standard DNA regions, facilitates prompt and accurate identification of traditional medicinal plants. The process of DNA barcoding entails six basic steps: 1) processing the medicinal plants, 2) extracting high-quality total DNA from the medicinal plants using centrifugal column method, 3) amplifying target DNA region internal transcribed spacer 2 (ITS2) with universal primers of plants and performing Sanger sequencing, 4) splicing and aligning sequence to obtain the target sequence, 5) matching the barcode sequence against the barcode library for identification, 6) aligning sequence, comparing intraspecific and interspecific variation, constructing phylogenetic neighbor-joining tree. As shown in the results, the universal primer can amplify the target region. Basic Local Alignment Search Tool (BLAST) demonstrates the percentage identified was 100%, and the neighbor-joining tree demonstrates that the splicing sequences were clustered with the A. sinensis OR879715.1 clade, and the clade support value is 100. This protocol provides a reference for applying DNA barcoding technology as an effective method to identify medicinal plants and adulterants.

Author Spotlight: Harnessing DNA Barcode Technology to Enhance the Efficiency of Medicinal Plant Identification

Application of DNA Barcoding to Identify Medicinal Plants

My research seeks to harness the power of DNA barcoding technology to enhance the accuracy and the efficiency of medical plant identification, thereby contributing to the preservation and the sustainable use of their valuable nature resource. DNA barcode technology has found broader applications such as quality control by monitoring or protect species and biodiversity assessment. High-throughput screening and high-throughput amplification are combined with DNA barcodes for species identification, which has expanded the application of herb identification based on DNA barcoding and brought out the past DNA barcoding error.Currently, there is a lack of comprehensive guidelines on how to identify medical plants and our process can provide detailed references. The primary research question for our laboratory in the future will focus on integrating DNA barcoding technology with technologies in our fields for broadening application and technology innovation.

Sample DNA quality
The range of OD260/OD280 absorbance ratios was 1.80-1.84. The DNA quantity measured by spectrophotometer for each sample was greater than 100 ng/&#181;L (Table 3), indicating a good quality of sample DNA extraction. This indicates that the samples were not contaminated by protein, RNA, or reagents during the extraction process and that they were of good quality and suitable for downstream PCR amplification.
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The present protocol describes the use of DNA barcoding technology to authenticate plant-based medicinal materials, and the medicinal plant Angelica sinensis (Oliv.) Diels was identified as an example.

Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration ...

application-of-dna-barcoding-to-identify-medicinal-plants

Research

JoVE Journal

Genetics

Extraction of DNA from Medicinal Plants for Species Verification Through DNA Barcoding

To begin, take 100 milligrams of fresh tissue or 20 milligrams of air-dried tissue and place it in a two-milliliter centrifuge tube. Place two five-millimeter stainless steel beads into the centrifuge tube and transfer it to a high-throughput tissue grinder operating at 60 hertz for 60 seconds. For DNA extraction, add 800 microliters of pre-cooled nuclear isolation buffer to the sample and place it on a vortex mixer for five minutes.Then, place the sample in a centrifuge and spin at 13, 780g for 10 minutes. After centrifugation, remove the supernatant carefully. Next, add 600 microliters of Buffer P1, followed by five microliters of RNase A to the sample.After vortexing, incubate the samples in a water bath at 65 degrees Celsius for 1.5 hours, inverting the tubes two to three times during incubation. Then, add 200 microliters of Buffer P2.Mix thoroughly. And place the sample on ice for 15 minutes.Afterward, centrifuge at 13, 780g for 10 minutes. Carefully aspirate the supernatant onto a filter column AF and centrifuge it at 13, 780g for two minutes. Transfer the lower filtrate to a new 1.5-milliliter centrifuge tube.After calculating the volume of the filtrate, add 1.5 times the volume of Buffer P3 to the sample and shake immediately to mix the sample. Add 650 microliters of the prepared mixture to an absorption column and incubate for five minutes. Then, centrifuge at 13, 780g for 30 to 60 seconds.Add the obtained filtrate into the absorption column again. Centrifuge and discard the waste liquid. Wash the column twice with 600 microliters of rinse solution WB.After centrifugation, discard the waste liquid, then place the absorption column into an empty collection tube.After centrifuging the sample at 13, 780g for two minutes, leave it at room temperature to evaporate residual ethanol. Transfer the absorption column into a clean 1.5-milliliter centrifuge tube, and add 45 microliters of sterilized deionized water preheated to 65 degrees Celsius to the center of the adsorbent membrane. Five minutes later, centrifuge the tube at 13, 780g for two minutes at room temperature.Then, use a spectrophotometer. Determine the DNA concentration in the filtered solution. The absorbance ratios of OD260 to OD280 ranged from 1.8 to 1.84, and the DNA quantity exceeded 100 nanogram per microliter, confirming good quality of the extracted DNA.

DNA Amplification, Sequencing, and Data Analysis to Apply DNA Barcoding for the Authentication of Medicinal Plants

To begin, extract DNA from the plant tissue and determine the DNA concentration. Set up the reaction after adding all the required components to a 0.2 milliliter centrifuge tube. After the primer amplification, load three microliters of the PCR product and two microliters of a 5, 000 base pair DNA marker to the wells of an agarose gel.Set the voltage to 120 volts, the current to 400 milliamps, and run the electrophoresis for 40 minutes. Use a versatile gel image analysis system to view and analyze the gel results. After running the sample through a sequencing unit, select Create a New Project and click Okay to go to the software homepage.Under the File menu, select Import and Add Samples. Choose the sequence trace format file and import it as the file to be processed under unassembled samples. Choose Contig.Then go to Advanced Assembly and select Assemble in Groups. When the dialogue prompting to define names appears, modify the file name in the define sample name parts section, and click Assemble to display the aligned sequence. Select Go, then choose Search Sequence and click Okay to find the matching sequence.Choose Contig, then select Delete to remove the 5.8S and 28S regions, along with any low quality sequences. Export the output with the spliced sequences for matching. Select a BLAST search using the Nucleotide Database at NCBI.Choose the species identification tool and the specific primer ITS-2, corresponding to the Global Pharmacopoeia Genome Database. Download sequences of three species of Angelica and one species of Peucedanum praeruptorum as an outgroup from NCBI. Analyze these sequences along with the result sequences obtained.Then click File. Select Open a File or Session to import the file and a dialogue will appear. Choose Align followed by DNA and select Align by ClustalW for sequence comparison.Navigate to phylogeny and click Construct Test Neighbor Joining Tree to generate a phylogenetic tree. Gel electrophoresis results showed single clear bands around 500 base pairs for samples AS1, AS2, and AS3, with no bands in the negative control, indicating successful amplification of the extracted DNA. Sequencing results identified Angelica sinensis with 100%sequence similarity using BLAST, matching results from the GPGD database.In the phylogenetic analysis, the splicing sequences clustered with Angelica sinensis OR8797150.1, with a bootstrap support value of 100, proving the identification as Angelica sinensis.

330 Views.  Guizhou University of Traditional Chinese Medicine.  The present protocol describes the use of DNA barcoding technology to authenticate plant-based medicinal materials, and the medicinal plant Angelica sinensis (Oliv.) Diels was identified as an example.

DNA Barcoding for Medicinal Plants: Identification and Phylogenetic Analysis

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