Method for identifying rhizoma cibotii medicinal material base and adulterants of rhizoma cibotii medicinal material base based on rbcL bar code
By applying rbcL barcode technology in the identification of dog spine medicinal materials, the systematic evolution tree and K2P genetic distance analysis was constructed, and the problem of difficulty in accurately identifying dog spine medicinal materials and their mixed products in the existing technology was solved, and the high-accurate identification results were achieved, ensuring the safety and standardization of the medicinal materials.
Patent Information
- Application Number
- CN202411887425.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-20
- Publication Date
- 2025-03-21
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art is difficult to accurately and quickly identify dog spine medicinal materials and their mixed counterfeit products, resulting in a large number of counterfeit products on the market, affecting the safety and standardization of clinical medicines of medicinal materials.
Using rbcL barcode technology, the genomic DNA of the sample to be tested was extracted, the rbcL gene was used as a molecular marker for PCR amplification and sequencing, and the phylogenetic tree and K2P genetic distance analysis was constructed to identify dog spine medicinal materials and mixed products.
It has achieved accurate identification of dog spine medicinal materials and their mixed products, and can clearly distinguish species from different sources, improving the accuracy and safety of medicinal materials.
Smart Images
Figure CN119662880A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of molecular identification of medicinal material origins, and more particularly to a molecular identification method of Cibotium barometz medicinal material origins and their adulterants. Background Art
[0002] The medicinal material Cibotium barometz is the dried rhizome of the large terrestrial fern plant Cibotium barometz, which has been included in the Pharmacopoeia of the People's Republic of China (2020 Edition) . Cibotium barometz is mostly wild and distributed in sunny mountain slopes and valleys in countries such as China, Vietnam, Japan and Indonesia. The villi on the surface of the rhizome of Cibotium barometz are multicellular trichomes, which can be used as a hemostatic agent for external application. For example, the dehaired rhizome is cut into slices and dried, which can be used to treat low back pain, rheumatism, sciatica, etc. Modern pharmacological and clinical studies have shown that the homogeneous polysaccharides (CBP70-1-1 and CBP70-1-2) in the rhizome extract of Cibotium barometz promote bone formation by activating the BMP2 / SMAD1 signaling pathway in MC3T3-E1 cells, and can be used as a natural anti-osteoporosis natural agent. In addition, the rhizome extract of Cibotium barometz also has pharmacological effects such as anti-oxidation, stimulation of chondrocyte proliferation and prevention of gastric ulcers. Therefore, the market demand for the medicinal material Cibotium barometz is increasing.
[0003] However, Cibotium barometz has a long growth cycle and a low reproduction rate, and its harvesting speed is far slower than its growth speed. It has now been listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Due to its significant therapeutic effect and insufficient supply, a large number of Cibotium barometz counterfeits have appeared on the market. Many plants of the same family or plants with similar morphology are sold as genuine Cibotium barometz slices, such as Adiantum raddianum, Woodwardia unigemmata, Phlebodium aureum, and Pteris wallichiana.
[0004] Traditional identification methods for Cibotium barometz and its counterfeits mainly rely on properties, microscopy and chemical identification methods. Property identification is mainly based on the characteristics of roots, leaves, spore groups, etc. to identify the authenticity of counterfeits. However, this method has great disadvantages. If the sample morphology is incomplete, it is impossible to identify the specific species. The authenticity of large-diameter Cibotium barometz slices circulating on the market can be identified based on the number of columnar vascular bundles, while it is more difficult to distinguish small-diameter slices. In addition, some researchers used thin-layer chromatography and kaempferol as a reference to identify Cibotium barometz and its counterfeits, but the versatility and accuracy of this method are difficult to estimate. Afterwards, the researchers further tried methanol ultrasonic extraction, ethyl acetate extraction, and a variety of development systems, such as n-butanol-acetic acid-water (3:2:1), chloroform-methanol-water (6.5:3.5:0.5) system, etc., but the development effect has problems such as fewer characteristic spots or heavy background interference, and it is still impossible to identify Cibotium barometz and its counterfeits.
[0005] In view of the limitations of identification of traits, microscopic and physicochemical properties, the establishment of an accurate and rapid DNA barcode identification method for the medicinal material Cibotium barometz and its adulterants is of great significance to the safety and standardization of the clinical use of Cibotium barometz. Summary of the invention
[0006] In view of the above problems, the present invention applies rbcL barcode technology to the identification of Cibotium barcode origin and its adulterants, and verifies the effectiveness of rbcL in identifying Cibotium barcode.
[0007] In order to achieve the above object, the present invention adopts the following technical solution:
[0008] A method for identifying the original source of Cibotium barcode and its mixed and counterfeited products based on rbcL barcode comprises the following steps:
[0009] (1) extracting genomic DNA of the sample to be tested, and using it as a template, performing PCR amplification using primers with the rbcL gene of chloroplast DNA as a molecular marker, performing bidirectional sequencing on the PCR amplification product, and calibrating the sequencing results by sequence splicing to obtain an accurate rbcL sequence as a comparison sequence, wherein the sequence of the upstream primer of the rbcL gene is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2;
[0010] (2) Perform BLAST comparison on all sequences obtained in step (1) in the NCBI database, determine the source species based on the homologous gene with the highest similarity score and the lowest E value, and download the reference sequence;
[0011] (3) aligning and trimming the comparison sequence obtained in step (1) and the reference sequence obtained in step (2), analyzing the inter-species and intra-species K2P genetic distances and constructing a phylogenetic tree;
[0012] (4) If the phylogenetic tree of the gene sequences of the samples to be tested clusters into the same species, then the samples to be tested are of that species.
[0013] Preferably, in step (1), the upstream primer sequence of the rbcL gene shown in SEQ ID NO:1 is specifically 5'-ATGTCACCACAAACAGAAACTAAAGC-3', and the upstream primer sequence of the rbcL gene shown in SEQ ID NO:2 is specifically 5'-TCGCATGTACCTGCAGTTGC-3'.
[0014] Preferably, the PCR amplification system described in step (1) is: 15 μl of 2×Taq PCR master mix, 1 μl of each of the upstream and downstream primers with a concentration of 10 μmol / L, 1 μl of genomic DNA with a concentration of 100 ng / μL, and 12 μl of ddH2O supplemented to 30 μl.
[0015] Preferably, the PCR amplification procedure in step (1) is: pre-denaturation at 95°C for 4 min; denaturation at 94°C for 45 s, annealing at 54°C for 1 min, extension at 72°C for 1 min, for a total of 35 cycles; and finally extension at 72°C for 10 min and storage at 4°C.
[0016] Preferably, the stitching calibration in step (1) specifically comprises: stitching and calibrating the sequences completed by the bidirectional sequencing using SeqMAN and DNAMAN software under the Lasergene software package, and removing low-quality sequences and primer regions.
[0017] Preferably, step (3) includes: performing sequence alignment based on MAFFT, trimming the aligned sequences using the trimAl tool, calculating the inter-species and intra-species K2P genetic distances using MEGA7.0.26 software, checking the support rate of each branch using the bootstrap method repeated 1000 times, and constructing a phylogenetic tree based on model selection using ModelTest-NG and Akaike information criterion and RAxML-NG software.
[0018] Preferably, the method for constructing the phylogenetic tree based on the rbcL gene in step (3) is the maximum likelihood method.
[0019] Through the above technical scheme, it can be known that the present invention applies rbcL barcode technology to the identification of Cibotium barometz and its mixed and counterfeit products. By collecting Cibotium barometz and its mixed and counterfeit products from different origins, the rbcL sequence of Cibotium barometz species is constructed in combination with the mixed and counterfeit product sequences downloaded from GenBank, and rbcL is determined to be the best DNA barcode for Cibotium barometz by using the phylogenetic tree based on rbcL barcode and K2P genetic distance analysis screen.
[0020] The present invention discloses a method for identifying the origin of Cibotium barcode-based medicinal materials and its adulterated products. By using rbcL gene amplification primers to perform PCR amplification on the DNA sequence of the sample to be tested to obtain the corresponding sequence, an evolutionary tree and a barcode are established for the obtained sequence matrix, species can be clearly separated, and the source species of Cibotium barcode-based medicinal materials and its adulterated products can be clearly distinguished, which provides a new idea for the identification of the origin of Cibotium barcode-based medicinal materials and its adulterated products, and is of great significance for solving the problem of adulteration of Cibotium barcode-based medicinal materials in the medicinal materials market. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 : A phylogenetic tree of golden retrievers and their hybrids constructed using rbcL sequences;
[0022] Figure 2 :Phylogenetic tree of golden retriever and its hybrids constructed using psbA-trnH sequences. DETAILED DESCRIPTION
[0023] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0024] The reagents involved in the embodiments of the present invention are all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.
[0025] FastPure Plant DNA Isolation Mini Kit Polysaccharide and Polyphenol Plant DNA Extraction Kit (DC104-01), Gel DNA Extraction Mini Kit (DC301-01) and 2×Taq PCR master mix (P112-01) were purchased from Nanjing Novozymes Biotech Co., Ltd.
[0026] 1×TAE buffer (B040123), 4S Green Plus nucleic acid dye (A616696), agarose (A620014), and anhydrous ethanol (A620014) were purchased from Sangon Biotech (Shanghai) Co., Ltd.
[0027] Color-changing silica gel (HG / T2765.4-2005) was purchased from Qingdao Ocean Chemical Co., Ltd.
[0028] Materials and Methods
[0029] 1. Sample collection and specimen identification
[0030] Field sampling of Cibotium barometz and plants that are easily confused with product sources was carried out in Hengzhou, Nanning, Laibin, Hechi, Guilin, Wuzhou and Lipu of Guangxi Zhuang Autonomous Region. The distance between all sampled individuals of the same population was greater than 50m. A total of 76 fresh leaves were collected, including Cibotium barometz (numbered Cb001 to Cb024), Adiantum raddianum (numbered Ar001 to Ar018), Woodwardia unigemmata (numbered Wu001 to Wu015), Phlebodium aureum (numbered Pa001 to Pa013), and Pteris wallichiana (numbered Pw001 to Pw006). The collected fresh leaves were immediately placed in a sealed sample bag containing sufficient silica gel (to avoid DNA degradation) for later use. All research related to the collected samples was approved by the Guangxi Zhuang Autonomous Region Institute of Traditional Chinese Medicine, and the plant species were jointly identified by Chief Pharmacist Huang Yunfeng and Deputy Chief Pharmacist Yan Kejian of the Guangxi Zhuang Autonomous Region Institute of Traditional Chinese Medicine based on traditional morphological methods. The specific collection locations are shown in Table 1. The voucher samples and digital image information are kept in the Guangxi Zhuang Autonomous Region Institute of Traditional Chinese Medicine Herbarium.
[0031] Table 1 Collection information of golden retrievers and their counterfeits
[0032]
[0033]
[0034]
[0035] 2. DNA extraction, amplification, sequencing and sequence assembly
[0036] (1) Take out silica gel dried leaves from each sample, freeze them quickly with liquid nitrogen, grind them into powder in a mortar, and take 50 mg of each leaf. Use FastPure Plant DNA Isolation Mini Kit Polysaccharide and Polyphenol Plant DNA Extraction Kit (Vazyme, DC104-01) to extract genomic DNA (water bath time is adjusted to 120 min, other specific operations refer to the corresponding instructions of the kit). Measure the quality and concentration of genomic DNA extracted from each sample using a spectrophotometer (NanoDrop 1000, Thermo Fisher). Dilute or concentrate the DNA solution of each sample to about 100 ng / μL for later use.
[0037] (2) PCR amplification was performed using primers rbcL-F (5'-ATGTCACCACAAACAGAAACTAAAGC-3', SEQ ID No. 1) and rbcL-R (5'-TCGCATGTACCTGCAGTTGC-3', SEQ ID No. 2) and the DNA solutions of the above samples (100 ng / μL) as templates. The PCR amplification system was as follows: 15 μl of 2×Taq PCR mastermix, 1 μl of each upstream and downstream primer at a concentration of 10 μmol / L, 1 μl of genomic DNA at a concentration of 100 ng / μL, and 12 μl of ddH2O to supplement the system to 30 μl. The PCR reaction procedure was as follows: pre-denaturation at 95°C for 4 min; denaturation at 94°C for 45 s, annealing at 54°C for 1 min, and extension at 72°C for 1 min, for a total of 35 cycles; and finally extension at 72°C for 10 min, followed by storage at 4°C.
[0038] (3) The products obtained by the PCR amplification were bidirectionally sequenced by Sangon Biotechnology (Shanghai) Co., Ltd. The peaks obtained after sequencing were proofread and spliced using SeqMAN and DNAMAN software under the Lasergene software package to remove low-quality sequences and primer regions to obtain the accurate rbcL sequence, as shown in Table 2.
[0039] Table 2 Basic information of rbcL sequences of golden retriever and its counterfeits
[0040]
[0041]
[0042]
[0043] 3. Characteristic comparison and genetic analysis
[0044] (1) The rbcL sequences shown in Table 2 were used as seeds to search for homologous sequences in the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) by BLAST. The source species was determined based on the homologous gene with the highest similarity score and the lowest E value. The rbcL sequences of five species were downloaded from GenBank, which were used as reference sequences. The specific information is shown in Table 3.
[0045] Table 3 Homologous rbcL sequences in NCBI database
[0046]
[0047]
[0048] (2) Genetic analysis was performed on the rbcL sequences shown in Tables 1 and 2. Specifically, sequence alignment was performed based on MAFFT, the trimAl tool was used to trim the aligned sequences, the inter-species and intra-species K2P genetic distances were calculated using MEGA7.0.26 software, and the support rate of each branch was checked using the bootstrap method (1000 repetitions). The results are shown in Table 4.
[0049] Table 4 Intra- and inter-species K2P genetic distances of rbcL sequences of golden retriever and its hybrids
[0050]
[0051]
[0052] As shown in Table 4, under the rbcL sequence, the average K2P distance within the species of Adiantum sphenanthera was 0.866, and the interspecific K2P distances between it and Adiantum sphenanthera, Cibotium apical, Polypodium cyanum and Pteris sphenanthera were 42.674, 32.458, 42.917 and 36.458, respectively. The average K2P distance within the species of Adiantum sphenanthera was 0.444, and the interspecific K2P distances between it and Adiantum sphenanthera, Cibotium apical, Polypodium cyanum and Pteris sphenanthera were 42.674, 32.458, 42.917 and 36.458, respectively. Similarly, the average K2P distances within the five species were smaller than the interspecific K2P distances between this species and the other four species.
[0053] The maximum likelihood method was used to construct a phylogenetic tree based on rbcL sequences in MEGA7.0.26 software. The bootstrap method of 1000 repetitions of tree construction was used to test the support rate of each branch. The results are shown in Figure 1 .
[0054] Depend on Figure 1 It can be seen that based on the rbcL barcode, the five species mainly formed four groups, group I was Pteris dasyphylla; group II was Adiantum cuneiformis; group III included Cibotium apical bud and Polypodium cyanobacteria, indicating that Cibotium apical bud and Polypodium cyanobacteria were closely related and clustered into one branch; group IV was Cibotium chinense, which clustered into one branch alone. The species can be clearly separated, and the source species of Cibotium chinense and its mixed and counterfeit products can be clearly distinguished, indicating that rbcL barcode can be used for molecular identification of Cibotium chinense medicinal materials and their mixed and counterfeit products.
[0055] Comparative Example
[0056] The psbA-trnH barcode is used to identify the original source of Cibotium barometz and its mixed products. The specific process is as follows:
[0057] (1) Using the DNA of each sample collected from the field for extracting Cibotium wilfordii and the source plants of easily confused products as templates, and using psbA-F (5'-GTTATGCATGAACGTAATGCTC-3', SEQ ID No. 3) and psbA-R (5'-TCGCATGTACCTGCAGTTGC-3', SEQ ID No. 4) as primers, PCR amplification was performed, wherein the PCR amplification system was: 15 μl of 2×Taq PCR master mix, 1 μl of each upstream and downstream primer with a concentration of 10 μmol / L, 1 μl of genomic DNA with a concentration of 100 ng / μL, and 12 μl of ddH2O to make up to 30 μl; the PCR reaction program was: pre-denaturation at 95°C for 4 min; denaturation at 94°C for 45 s, annealing at 55°C for 45 s, and extension at 72°C for 45 s, for a total of 35 cycles; and finally extension at 72°C for 10 min, and storage at 4°C.
[0058] (2) The products obtained by the PCR amplification were bidirectionally sequenced by Sangon Biotechnology (Shanghai) Co., Ltd. The peaks obtained after sequencing were proofread and spliced using SeqMAN and DNAMAN software under the Lasergene software package to remove low-quality sequences and primer regions to obtain accurate psbA-trnH sequences, as shown in Table 5.
[0059] Table 5 Basic information of psbA-trnH sequences of golden retrievers and their counterfeits
[0060]
[0061] (3) The psbA-trnH sequences shown in Table 5 were used as seeds to search for homologous sequences in the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) by BLAST. The source species was determined based on the homologous gene with the highest similarity score and the lowest E value. The psbA-trnH sequences of five species were downloaded from GenBank, which were used as reference sequences. The specific information is shown in Table 6.
[0062]
[0063]
[0064] (4) Genetic analysis was performed on the psbA-trnH sequences shown in Tables 5 and 6. Specifically, sequence alignment was performed based on MAFFT, the trimAl tool was used to trim the aligned sequences, and the MEGA7.0.26 software was used to calculate the inter-species and intra-species K2P genetic distances. The results are shown in Table 7.
[0065] Table 7 Intra- and inter-species K2P genetic distances of psbA-trnH sequences of golden retrievers and their hybrids
[0066]
[0067]
[0068] As shown in Table 7, under the psbA-trnH sequence, the average K2P distance within the species of P. chinensis was 0.000, and the interspecific K2P distances between it and Adiantum cuneiformis, Cibotium apical, Polypodium cyanum and Pteris suspensa were 220.000, 215.625, 221.250 and 189.250, respectively. The average intraspecific K2P distances of the five species were all smaller than the interspecific K2P distances between this species and the other four species.
[0069] The maximum likelihood method was used to construct a phylogenetic tree based on the psbA-trnH sequence in MEGA7.0.26 software. The bootstrap method of 1000 repetitions of tree construction was used to test the support rate of each branch. The results are shown in Figure 2 .Depend on Figure 2 It can be seen that based on the psbA-trnH barcode, the five species cannot be clustered in a single branch, indicating that the psbA-trnH barcode cannot be used for the molecular identification of the original source of Cibotium barometz and its adulterants.
[0070] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.
[0071] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. Application of rbcL barcode in identification of the origin of Cibotium barometz and its adulterants.
2. A method for identifying the original source of Cibotium barcode and its counterfeit products based on rbcL barcode, characterized in that: The following steps are included: (1) extracting genomic DNA of the sample to be tested, and using it as a template, performing PCR amplification using primers with rbcL gene of ribosomal DNA as a molecular marker, performing bidirectional sequencing on the PCR amplification product, and calibrating the sequencing results by sequence splicing to obtain an accurate rbcL sequence as a comparison sequence, wherein the sequence of the upstream primer of the rbcL gene is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2; (2) Perform BLAST comparison on all sequences obtained in step (1) in the NCBI database, determine the source species based on the homologous gene with the highest similarity score and the lowest E value, and download the reference sequence; (3) aligning and trimming the comparison sequence obtained in step (1) and the reference sequence obtained in step (2), analyzing the inter-species and intra-species K2P genetic distances and constructing a phylogenetic tree; (4) If the phylogenetic tree of the gene sequences of the samples to be tested clusters into the same species, then the samples to be tested are of that species.
3. The method for identifying the original source of Cibotium barcode and its counterfeit products based on rbcL barcode according to claim 2, characterized in that: The PCR amplification system described in step (1) is: 2×Taq PCR master mix 15 μl, 1 μl of upstream and downstream primers with a concentration of 10 μmol / L, 1 μl of genomic DNA with a concentration of 100 ng / μL, and 12 μl ddH2O supplemented to 30 μl.
4. The method for identifying the original source of Cibotium barcode and its mixed and counterfeited products based on rbcL barcode according to claim 2, characterized in that: The PCR amplification procedure described in step (1) is: pre-denaturation at 95°C for 4 minutes; denaturation at 94°C for 45 seconds, annealing at 54°C for 1 minute, extension at 72°C for 1 minute, for a total of 35 cycles; and finally extension at 72°C for 10 minutes, and storage at 4°C.
5. The method for identifying the original source of Cibotium barcode and its counterfeit products based on rbcL barcode according to claim 2, characterized in that: The stitching calibration in step (1) specifically includes: using SeqMAN and DNAMAN software under Lasergene software package to stitch and calibrate the sequences completed by the bidirectional sequencing, and removing low-quality sequences and primer regions.
6. The method for identifying the original source of Cibotium barcode and its counterfeit products based on rbcL barcode according to claim 2, characterized in that: Step (3) included: sequence alignment based on MAFFT, trimming the aligned sequences using the trimAl tool, calculating the inter- and intra-species K2P genetic distances using MEGA7.0.26 software, checking the support rate of each branch using the bootstrap method repeated 1000 times, and constructing a phylogenetic tree based on model selection using ModelTest-NG and the Akaike information criterion and RAxML-NG software.
7. The method for identifying the original source of Cibotium barcode and its adulterated products based on rbcL barcode according to claim 2, characterized in that: The method for constructing the phylogenetic tree based on the rbcL gene in step (3) is the maximum likelihood method.