Method for identifying three varieties of Polygonatum officinale

Abstract

The present invention belongs to the technical field of traditional Chinese medicine quality control, and specifically discloses a method for identifying three varieties of medicinal Polygonatum: the three varieties of medicinal Polygonatum are Polygonatum kingianum Coll. et Hemsl., Polygonatum cyrtonema Hua, and Polygonatum sibiricum Redoute. Taking the tuber of the Polygonatum to be tested as a sample, the sample is extracted, and the metabolites in the sample are detected by liquid chromatography-mass spectrometry. Then, the metabolites are compared with the characteristic metabolites of Polygonatum kingianum Coll. et Hemsl., Polygonatum cyrtonema Hua, and Polygonatum sibiricum Redoute respectively, so as to identify the variety of Polygonatum. When the metabolites detected in the sample belong to the characteristic metabolites of a certain variety of Polygonatum, it is determined that the sample is the variety of Polygonatum corresponding to the characteristic metabolites. The present invention provides a scientific basis for the quality identification and quality control of the three varieties of medicinal Polygonatum, and helps to quickly and accurately identify the authenticity and quality of the commercially available medicinal Polygonatum.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine quality control, and particularly relates to a method for identifying three medicinal polygonatum varieties. Background Art

[0002] Polygonatum sibiricum is a traditional plant used for both medicine and food. Studies have shown that the rhizome of Polygonatum sibiricum does not contain starch, but is rich in nutrients and medicinal functional ingredients such as polysaccharides, oligofructose, flavonoids, and triterpenoid saponins. The 2020 edition of the Chinese Pharmacopoeia includes three source species of Polygonatum sibiricum, P. kingianum, and P. cyrtonema, which have the effects of replenishing qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. Polygonatum sibiricum is suitable for large-scale planting under forests in subtropical, temperate, and cold temperate zones, and is an emerging high-quality grain resource with great potential that does not occupy farmland.

[0003] The appearances of Polygonatum sibiricum, Polygonatum yunnanensis and Polygonatum cyrtonema are quite similar, so it is difficult to distinguish them based on their appearance alone.

[0004] The current detection methods are as follows:

[0005] The metabolites in medicinal polygonatum were analyzed and detected by gas chromatography-mass spectrometry; the differential metabolites of three medicinal polygonatum were screened by multivariate statistical methods, for example:

[0006] 1. The invention of CN111505171A, "A method for identifying three varieties of medicinal polygonatum", provides a method for identifying three varieties of medicinal polygonatum, which belongs to the technical field of quality control of traditional Chinese medicine. The invention extracts and derivatizes the three varieties of medicinal polygonatum, analyzes and detects the metabolites in the medicinal polygonatum by gas chromatography-mass spectrometry, and screens the differential metabolites of the three varieties of medicinal polygonatum by multivariate statistical methods. The differential metabolites are used as characteristic metabolites for identifying the three varieties of medicinal polygonatum, thereby realizing the identification of the three varieties of medicinal polygonatum.

[0007] When performing the gas chromatography-mass spectrometry analysis, the operating conditions of the gas chromatography include:

[0008] DB-5 quartz capillary column 60m×250μm×0.25μm; split injection, injection volume 1μL, split ratio 10:1; injection port temperature 280℃; ion source temperature 250℃; interface temperature 250℃; heating program: initial temperature 40℃, hold for 5min, heat to 280℃ at 8℃ / min, hold for 5min; carrier gas is helium, carrier gas flow rate 1mL / min; solvent delay time 14min; mass detection range in terms of m / z is 33-600;

[0009] The operating conditions of the mass spectrometer include:

[0010] Electrospray ionization source, ion source temperature is 230 °C, quadrupole temperature is 150 °C, full scan mode, electron energy is 70 eV; in terms of m / z, the quadrupole scanning range is 35 - 780.

[0011] 2. The invention of CN108845071A, "Identification Method of Three Kinds of Legitimate Source Polygonatum Sibiricum", relates to the identification method of three kinds of legitimate source Polygonatum Sibiricum. The medicinal materials collected are extracted by ultrasonic method. After the extract is purified by PS / AL solid phase extraction column, the chemical components are characterized by ultra-high performance liquid chromatography tandem electrostatic field orbitrap high resolution mass spectrometry (UPLC-Orbitrap MS) technology, and the data is processed by multivariate statistical analysis, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares analysis (OPLS-DA) and hierarchical cluster analysis (HCA).

[0012] UHPLC / MS analysis:

[0013] The UHPLC conditions are as follows: chromatographic column: Agilent Zorbax SB-C18; mobile phase: gradient elution of acetonitrile A and water B: 0 min, 10% A; 20 min, 25% A; 30 min, 35% A; 40 min, 50% A; 50 min, 65% A; 60 min, 85% A; 70 min, 100% A; 75 min, 100% A; flow rate 1.0 mL / min; column temperature 30 °C; injection volume 10 μL;

[0014] The mass spectrometry conditions are as follows: detection is carried out in full scan and data-dependent MS / MS mode; the ion source is ESI; capillary temperature 320 °C; spray gas flow rate 1.5 L / min; heating module 250 °C; full scan positive and negative ions are detected simultaneously, resolution 70,000; interface voltage: (+) 3.5 kV, (-) 2.8 kV; mass range m / z: 100 - 1000 Da; maximum injection time: 200 msec; MS / MS positive and negative ions are detected simultaneously, data-dependent MS / MS performs MS / MS fragmentation on the top 5 peaks: resolution 17,500, mass range m / z: 50 - 1000 Da; maximum injection time: 50 msec; CID energy: 30%; dynamic exclusion is used, dynamic exclusion time: 10 s; workstation: Xcalibar 3.0.63;

[0015] The disadvantages of the above scheme are that the detection method of the main active substances in medicinal plants is detected by liquid chromatography-mass spectrometry coupling technology, and the substances detected by gas phase may not be representative, and the main active substances cannot be detected by gas chromatography-mass spectrometry; there are large differences in different metabolites among different germplasm resources, and this method does not have wide applicability. Summary of the Invention

[0016] The technical problem to be solved by the present invention is to provide a method for identifying medicinal Polygonatum sibiricum using liquid chromatography-mass spectrometry (LC-MS) technology.

[0017] To solve the above technical problem, the present invention provides a method for identifying three varieties of medicinal Polygonatum sibiricum, namely Polygonatum kingianum Coll. et Hemsl., Polygonatum cyrtonema Hua, and Polygonatum sibiricum Red.;

[0018] Taking the tuberous roots of the Polygonatum sibiricum to be tested as samples, the samples are extracted, and the metabolites in the samples are detected using liquid chromatography-mass spectrometry (LC-MS) technology; then, they are compared with the characteristic metabolites of Polygonatum kingianum Coll. et Hemsl., Polygonatum cyrtonema Hua, and Polygonatum sibiricum Red. respectively, so as to identify the variety of Polygonatum sibiricum.

[0019] As an improvement to the method for identifying three varieties of medicinal Polygonatum sibiricum of the present invention:

[0020] When the metabolite detected in the sample belongs to the characteristic metabolite of a certain variety of Polygonatum sibiricum, it is determined that the sample is the variety of Polygonatum sibiricum corresponding to the characteristic metabolite.

[0021] As a further improvement to the method for identifying three varieties of medicinal Polygonatum sibiricum of the present invention:

[0022] The characteristic metabolites of Polygonatum kingianum Coll. et Hemsl. are: delphinidin-3-O-glucoside (neg_2419), neomangiferin (neg_2461), shikonin (neg_7649);

[0023] The characteristic metabolites of Polygonatum cyrtonema Hua are: narirutin (neg_10553), daidzin (neg_7594), loureirin B (neg_9283), wilfordine (pos_2718), apigenin 7-O-neohesperidoside (pos_5290);

[0024] The characteristic metabolites of Polygonatum sibiricum Red. are: petunidin-3-O-[6”-O-(Z)p-coumaroyl rutinoside]-5-O-glucoside (neg_2293), geniposide triacetate (pos_10556), typhaneoside (pos_4885).

[0025] As a further improvement to the method for identifying three varieties of medicinal Polygonatum sibiricum of the present invention, the obtaining of the metabolites in the sample includes the following steps:

[0026] 1), Grind the tuberous roots of the Polygonatum sibiricum to be tested in liquid nitrogen, weigh 50 mg of the ground Polygonatum sibiricum tissue, add 1000 ± 50 μL of extraction solution, mix well and ultrasonicate (ultrasonic power is 50 W) for 10 ± 1 min to obtain a crude extract;

[0027] The extraction solution is an aqueous methanol - acetonitrile solution obtained by mixing methanol, acetonitrile, and water in a volume ratio of 2:2:1;

[0028] 2), The crude extract obtained in step 1) is left to stand at -20 ± 5 °C for 1 ± 0.1 h, and then centrifuged at 4 ± 1 °C (centrifuged at 12000 ± 2000 rpm for 15 ± 1 min) to obtain the supernatant;

[0029] 3), Take 500 μL of the supernatant obtained in step 2) and dry it (the drying temperature is about 35 °C until constant weight); add 160 μL of acetonitrile aqueous solution for dissolution;

[0030] The acetonitrile aqueous solution is obtained by mixing acetonitrile and water in a volume ratio of 1:1;

[0031] 4), The solution obtained in step 3) is first vortex - shaken and then placed in an ice - water bath for ultrasonic treatment (ultrasonic power is 50 W) for 10 ± 1 min; then centrifuged at 4 ± 1 °C (centrifuged at 12000 ± 2000 rpm for 15 ± 1 min); the obtained supernatant is used as the sample - loading solution;

[0032] 5), Take the sample - loading solution obtained in step 4) for LC - MS analysis; thereby, the metabolites in the sample can be known.

[0033] As a further improvement of the method for identifying three varieties of medicinal Polygonatum sibiricum in the present invention, step 5) is:

[0034] The sample - loading solution is subjected to metabolomics analysis using a liquid chromatography - mass spectrometry system composed of Waters Acquity I - Class PLUS ultra - high performance liquid chromatography and Waters Xevo G2 - XS QTOF high - resolution mass spectrometer. The chromatographic column used is Waters Acquity UPLC HSS T3 chromatographic column (1.8 μm, 2.1 * 100 mm); mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is 0.1% formic acid acetonitrile;

[0035] The injection volume of liquid chromatography is 1 μL, the elution flow rate is 400 μL / min, and the elution gradient is: 0 - 0.25 min, 98% phase A, 2% phase B; 0.25 - 10 min, the concentration of phase A decreases uniformly from 98% to 2%, and the concentration of phase B increases uniformly from 2% to 98%; 10 - 13 min, maintain 2% phase A, 98% phase B; 13 - 13.1 min, the concentration of phase A increases uniformly from 2% to 98%, and the concentration of phase B decreases uniformly from 98% to 2%; 13.1 - 15 min, elute with 98% phase A and 2% phase B;

[0036] The operating conditions of the mass spectrometry are as follows: The capillary voltage of the ion source (MSI) is set to 2500 V in the positive ion mode and -2000 V in the negative ion mode; the cone voltage is set to 30 V; the ion source temperature is 100 °C; the desolvation gas temperature is 500 °C; the backflush gas flow rate is 50 L / h; the desolvation gas flow rate is 800 L / h; the mass-to-charge ratio (m / z) acquisition range is 50 - 1200;

[0037] The relative expression levels of metabolites are obtained from the peak maps obtained by liquid chromatography - mass spectrometry.

[0038] Note: The mass spectrometry used is a Waters Xevo G2-XS QTof high-resolution mass spectrometer, which can perform primary and secondary mass spectrometry data acquisition in the MSe mode under the control of the acquisition software (MassLynx V4.2, Waters). In each data acquisition cycle, dual-channel data acquisition can be performed simultaneously for low collision energy and high collision energy. The low collision energy is 2 V, the high collision energy range is 10 - 40 V, and the scanning frequency is one mass spectrum per 0.2 seconds.

[0039] As a further improvement to the method for identifying three varieties of medicinal Polygonatum sibiricum in the present invention:

[0040] The relative expression levels of metabolites are obtained from the peak maps obtained by liquid chromatography - mass spectrometry through the following method: Metabolite analysis uses the raw data collected by MassLynx V4.2 software to perform data processing operations such as peak extraction and peak alignment through Progenesis QI software, and is identified based on the online METLIN database and public databases of Progenesis QI software. At the same time, theoretical fragment identification is performed, with the deviation of the precursor ion mass number within 10 ppm and the deviation of the fragment ion mass number within 20 ppm.

[0041] As a further improvement to the method for identifying three varieties of medicinal Polygonatum sibiricum in the present invention:

[0042] In step 1), an extraction solution containing an internal standard is used; the extraction solution containing an internal standard is prepared by adding L-2-chlorophenylalanine to a concentration of 20 mg / L in a methanol - acetonitrile - aqueous solution.

[0043] The present invention discloses a method for identifying three varieties of medicinal Polygonatum sibiricum. The three Polygonatum sibiricum are extracted, and the metabolites in Polygonatum sibiricum are detected by liquid chromatography - mass spectrometry (LC-MS) technology. By screening the specifically expressed metabolites in each variety of Polygonatum sibiricum as its characteristic metabolites, this is used as the basis for identifying different varieties of Polygonatum sibiricum. Based on the above, the method provided by the present invention can conveniently, efficiently, and accurately identify the varieties of Polygonatum sibiricum using the screened characteristic metabolites.

[0044] After the extraction and derivatization of three medicinal Polygonatum sibiricum Red., the metabolites in the medicinal Polygonatum sibiricum Red. were analyzed and detected by gas chromatography-mass spectrometry (GC-MS), and the differential metabolites of the three medicinal Polygonatum sibiricum Red. were screened by multivariate statistical methods. The differential metabolites were used as characteristic metabolites to identify the three varieties of medicinal Polygonatum sibiricum Red., so as to realize the identification of the three varieties of medicinal Polygonatum sibiricum Red. The method provided by the invention has high accuracy and reliability, and the experimental results are highly credible, providing a scientific basis for the quality identification and quality control of the three medicinal Polygonatum sibiricum Red., and helping to quickly and accurately identify the authenticity and quality of the commercially available medicinal Polygonatum sibiricum Red. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The following further describes the specific embodiments of the present invention in conjunction with the accompanying drawings.

[0046] Figure 1 It is a Venn diagram of the metabolite numbers of the three Polygonatum sibiricum Red.

[0047] Figure 1 In the figure, D represents Polygonatum kingianum Coll. et Hemsl., DH represents Polygonatum cyrtonema Hua, and H represents Polygonatum sibiricum Red.

[0048] Figure 2 It is a clustering diagram of metabolites in the three Polygonatum sibiricum Red.

[0049] Figure 3 It is a PCA diagram of metabolites in the three Polygonatum sibiricum Red.

[0050] Figure 4 It is a chromatogram of Polygonatum sibiricum Red.; the upper figure is in the positive ion mode, and the lower figure is in the negative ion mode.

[0051] Figure 5 It is a chromatogram of Polygonatum kingianum Coll. et Hemsl.; the upper figure is in the positive ion mode, and the lower figure is in the negative ion mode.

[0052] Figure 6 It is a chromatogram of Polygonatum cyrtonema Hua; the upper figure is in the positive ion mode, and the lower figure is in the negative ion mode. SPECIFIC EMBODIMENTS

[0053] The following further describes the present invention in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0054] The rotation speed of the vortex is 2000-3000 rpm.

[0055] Example 1. Method for obtaining characteristic metabolites in Polygonatum sibiricum Red., Polygonatum kingianum Coll. et Hemsl. and Polygonatum cyrtonema Hua:

[0056] Polygonatum sibiricum Red., Polygonatum kingianum Coll. et Hemsl. and Polygonatum cyrtonema Hua were used as test samples, and the following steps were carried out respectively first:

[0057] 1), After collecting various polygonatum rhizomes, grind them in liquid nitrogen. Accurately weigh 50 mg of the ground polygonatum tissue, add 1000 μL of the extraction solution containing an internal standard into a 1.5 ml EP tube (the extraction solution is a methanol-acetonitrile-water solution, with a volume ratio of methanol:acetonitrile:water of 2:2:1, and the internal standard is L-2-chlorophenylalanine with a concentration of 20 mg / L), and vortex for 30 seconds; sonicate in ice water (sonication power is 50 W) for 10 min to obtain a crude extract.

[0058] 2), Let the crude extract obtained in step 1) stand at -20 °C for 1 h, then set the centrifuge to centrifuge at 4 °C and a rotation speed of 12000 rpm for 15 min to obtain the supernatant.

[0059] 3), Take 500 μL of the supernatant obtained in step 2) into a new EP tube, and dry it to constant weight in a vacuum centrifugal concentrator (the drying temperature is about 35 °C); after drying, add 160 μL of acetonitrile-water solution (volume ratio of acetonitrile:water is 1:1) for dissolution.

[0060] 4), First vortex the solution obtained in step 3) for 30 s and then sonicate it in an ice-water bath (sonication power is 50 W) for 10 min; then centrifuge at 4 °C and a rotation speed of 12000 rpm for 15 min; the obtained supernatant is used as the sample loading solution.

[0061] 5), Take 120 μL of the sample loading solution obtained in step 4) into a 2 mL injection vial and then it can be used for subsequent on-machine detection, that is, perform LC-MS analysis on the sample loading solution;

[0062] Specifically as follows:

[0063] Perform metabolomics analysis on the said sample loading solution using a liquid chromatography-mass spectrometry system composed of Waters Acquity I-Class PLUS ultra-high performance liquid chromatography and Waters Xevo G2-XS QTOF high-resolution mass spectrometer. The chromatographic column used is Waters Acquity UPLC HSS T3 chromatographic column (1.8 um 2.1*100 mm). Mobile phase A is an aqueous solution of 0.1% formic acid by volume (0.1% formic acid + 99.9% water), and mobile phase B is acetonitrile with 0.1% formic acid by volume (0.1% formic acid + 99.9% acetonitrile). The injection volume of liquid chromatography is 1 μl, the elution flow rate is 400 (μL / min), and the elution gradient is: 0 - 0.25 min, 98% phase A, 2% phase B; 0.25 - 10 min, the concentration of phase A decreases uniformly from 98% to 2%, and the concentration of phase B increases uniformly from 2% to 98%; 10 - 13 min, maintain 2% phase A, 98% phase B; 13 - 13.1 min, the concentration of phase A increases uniformly from 2% to 98%, and the concentration of phase B decreases uniformly from 98% to 2%; 13.1 - 15 min, maintain 98% phase A, 2% phase B for elution.

[0064] The mass spectrometry was performed using a Waters Xevo G2-XS QTof high-resolution mass spectrometer, which was capable of collecting first- and second-order mass spectrometry data in the MSe mode under the control of the acquisition software (MassLynxV4.2, Waters). In each data acquisition cycle, dual-channel data acquisition could be performed simultaneously for both low and high collision energies. The low collision energy was 2V, the high collision energy range was 10 - 40V, and the scanning frequency was one mass spectrum per 0.2 seconds.

[0065] The operating conditions of the mass spectrometry were as follows: the capillary voltage of the ion source was set to 2500V in the positive ion mode and -2000V in the negative ion mode; the cone voltage was set to 30V; the ion source temperature was 100°C; the desolvation gas temperature was 500°C; the backflush gas flow rate was 50L / h; the desolvation gas flow rate was 800L / h; the mass-to-charge ratio (m / z) acquisition range was 50 - 1200.

[0066] The relative expression levels of metabolites were obtained by analyzing the peak maps obtained from liquid chromatography-mass spectrometry using the following method: metabolite analysis involved performing data processing operations such as peak extraction and peak alignment on the raw data collected by MassLynx V4.2 software using Progenesis QI software. Identification was based on the online METLIN database and public databases in Progenesis QI software, and theoretical fragment identification was also performed, with the mass deviation of the parent ion within 10ppm and the mass deviation of the fragment ion within 20ppm.

[0067] The metabolites unique to each polygonatum and those common to other polygonatums were counted by drawing Venn diagrams ( Figure 1 ). Among them, there were 76 metabolites unique to Polygonatum kingianum; 122 metabolites unique to Polygonatum cyrtonema; and 14 metabolites unique to Polygonatum sibiricum. Figure 2 This is the clustering analysis diagram of the three polygonatums. From Figure 2 it can be seen that there are differences in the metabolite compositions of the three polygonatums and they can be clearly distinguished. Figure 3 This is the principal component analysis (PCA) diagram of the metabolites of the three polygonatums. From Figure 3 it can be seen that the metabolite compositions of the three polygonatums can be clearly distinguished. Principal component 1 can explain 48.9% of the variation, and principal component 2 can explain 44.2% of the variation.

[0068] These unique metabolites were searched in the plant metabolite database to identify the metabolites listed in Table 1 as characteristic metabolites for differentiating the three medicinal polygonatums (unique to that polygonatum).

[0069] Table 1. List of characteristic metabolites in each polygonatum

[0070]

[0071]

[0072] Experiment 1. A method for identifying the variety of the Polygonatum odoratum to be tested, using the Polygonatum odoratum to be tested that is known in advance to be of the Polygonatum odoratum variety as a sample;

[0073] Steps 1) to 5) are the same as those in Example 1.

[0074] The chromatogram obtained from the liquid chromatography-mass spectrometry system (as Figure 4 described) The original data collected by importing the data into MassLynx V4.2 software is subjected to data processing operations such as peak extraction and peak alignment through Progenesis QI software. Identification is carried out based on the online METLIN database and public databases of Progenesis QI software, and at the same time, theoretical fragment identification is performed, with the deviation of the precursor ion mass number within 10 ppm and the deviation of the fragment ion mass number within 20 ppm. The metabolite expression information finally obtained shows that "neg_2293, pos_10556, pos_4885" can obtain corresponding information, while "neg_2419, neg_2461, neg_7649, neg_10553, neg_7594, neg_9283, pos_2718, pos_5290" cannot obtain corresponding information.

[0075] #ID H1 H2 H3 neg_2293 173.3574 166.9127 203.854 pos_10556 1085.92 1127.128 1161.326 pos_4885 76.12896 54.68045 89.45553

[0076] Compared with Table 1, it can be known that the Polygonatum odoratum to be tested belongs to the Polygonatum odoratum variety. It is completely consistent with the known situation in advance.

[0077] Experiment 2. A method for identifying the variety of the Polygonatum kingianum to be tested, using the Polygonatum kingianum to be tested that is known in advance to be of the Polygonatum kingianum variety as a sample;

[0078] Steps 1) to 5) are the same as those in Example 1.

[0079] The chromatogram obtained from the "liquid chromatography-mass spectrometry system" (as Figure 5The original data collected by importing the data into MassLynx V4.2 software was subjected to data processing operations such as peak extraction and peak alignment using Progenesis QI software. Identification was performed based on the online METLIN database and public databases of Progenesis QI software, and theoretical fragment identification was also carried out, with the deviation of the precursor ion mass number within 10 ppm and the deviation of the fragment ion mass number within 20 ppm. The metabolite expression information finally obtained showed that corresponding information could be obtained for "neg_2419, neg_2461, neg_7649", while no corresponding information could be obtained for "neg_10553, neg_7594, neg_9283, pos_2718, pos_5290, neg_2293, pos_10556, pos_4885".

[0080] #ID D1 D2 D3 neg_2419 766.1543 860.8089 800.4505 neg_2461 148.1594 129.6125 142.5497 neg_7649 184.5214 180.052 185.1824

[0081] Compared with Table 1, it can be known that the tested polygonatum belongs to the variety of Polygonatum kingianum Coll. et Hemsl. It is completely consistent with the previously known situation.

[0082] Experiment 3. A method for identifying the variety of the tested polygonatum, using the tested polygonatum that is known to be of the variety of Polygonatum cyrtonema Hua as a sample;

[0083] Steps 1) to 5) are the same as in Example 1.

[0084] The chromatogram obtained from the "liquid chromatography - mass spectrometry system" (as Figure 6 The original data collected by importing the data into MassLynx V4.2 software was subjected to data processing operations such as peak extraction and peak alignment using Progenesis QI software. Identification was performed based on the online METLIN database and public databases of Progenesis QI software, and theoretical fragment identification was also carried out, with the deviation of the precursor ion mass number within 10 ppm and the deviation of the fragment ion mass number within 20 ppm. The metabolite expression information finally obtained showed that corresponding information could be obtained for "neg_10553, neg_7594, neg_9283, pos_2718, pos_5290", while no corresponding information could be obtained for "neg_2419, neg_2461, neg_7649, neg_2293, pos_10556, pos_4885".

[0085] #ID DH-1 DH-2 DH-3 neg_10553 364.4744 574.7173 772.7851 neg_7594 339.026 341.322 363.8772 neg_9283 1477.324 1531.309 1577.247 pos_2718 122.2294 99.45698 135.9779 pos_5290 104.5558 115.938 101.1293

[0086] Compared with Table 1, it can be known that the tested polygonatum belongs to the variety of Polygonatum cyrtonema Hua. It is completely consistent with the previously known situation.

[0087] Experiment 4: The mixed samples of "Polygonatum sibiricum, Polygonatum yunnanensis and Polygonatum cyrtonema" were tested according to the method described in Experiment 1. The final metabolite expression information showed that: "neg_10553, neg_7594, neg_9283, pos_2718, pos_5290, neg_2419, neg_2461, neg_7649, neg_2293, pos_10556, pos_4885" could all obtain corresponding information.

[0088] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for identifying three varieties of medicinal Polygonatum, characterized in that: The three varieties of medicinal Polygonatum are Polygonatum kingianum (P. kingianum), Polygonatum cyrtonema (P. cyrtonema), and Polygonatum sibiricum; Using the tuberous roots of the Polygonatum to be tested as a sample, the sample is extracted, and the metabolites in the sample are detected by liquid chromatography-mass spectrometry; then compared with the characteristic metabolites of Polygonatum kingianum (P. kingianum), Polygonatum cyrtonema (P. cyrtonema), and Polygonatum sibiricum respectively, so as to identify the variety of Polygonatum; When the metabolites detected in the sample belong to the characteristic metabolites of a certain variety of Polygonatum, it is determined that the sample is the variety of Polygonatum corresponding to the characteristic metabolites; The characteristic metabolites of Polygonatum kingianum (P. kingianum) are: delphinidin-3-O-glucoside, neomangiferin, and shikonin; The characteristic metabolites of Polygonatum cyrtonema (P. cyrtonema) are: narirutin, daidzin, loureirin, wilfordine, and apigenin 7-O-neohesperidoside; The characteristic metabolites of Polygonatum sibiricum are: petunidin-3-O-[6''-O-(Z)p-coumaroylrutinoside]-5-O-glucoside, triptonide, and typhonoside; The acquisition of metabolites in the sample includes the following steps: 1). Grind the tuberous roots of the Polygonatum to be tested in liquid nitrogen, weigh 50 mg of the ground Polygonatum tissue, add 1000 ± 50 μL of the extraction solution, mix well and sonicate for 10 ± 1 min to obtain a crude extract; The extraction solution is an aqueous methanol-acetonitrile solution obtained by mixing methanol, acetonitrile, and water in a volume ratio of 2:2:1; 2). Let the crude extract obtained in step 1) stand at -20 ± 5 °C for 1 ± 0.1 h, then centrifuge at 4 ± 1 °C to obtain a supernatant; 3). Take 500 μL of the supernatant obtained in step 2) and dry it; add 160 μL of an acetonitrile aqueous solution for dissolution; The acetonitrile aqueous solution is obtained by mixing acetonitrile and water in a volume ratio of 1:1; 4). First vortex and shake the solution obtained in step 3), then place it in an ice bath and sonicate for 10 ± 1 min; then centrifuge at 4 ± 1 °C; the obtained supernatant is used as the sample loading solution; 5). Take the sample loading solution obtained in step 4) for LC-MS analysis to obtain the metabolites in the sample: The sample loading solution is analyzed by a liquid chromatography-mass spectrometry system composed of a Waters Acquity I-Class PLUS ultra-high performance liquid chromatography and a Waters Xevo G2-XS QTOF high-resolution mass spectrometer. The chromatographic column used is a Waters Acquity UPLC HSS T3 chromatographic column; mobile phase A is an aqueous solution of 0.1% formic acid by volume concentration, and mobile phase B is acetonitrile with 0.1% formic acid by volume concentration. The injection volume of liquid chromatography is 1 μL, the elution flow rate is 400 μL / min, and the elution gradient is as follows: from 0 to 0.25 min, 98% phase A and 2% phase B; from 0.25 to 10 min, the concentration of phase A decreases uniformly from 98% to 2%, and the concentration of phase B increases uniformly from 2% to 98%; from 10 to 13 min, 2% phase A and 98% phase B are maintained; from 13 to 13.1 min, the concentration of phase A increases uniformly from 2% to 98%, and the concentration of phase B decreases uniformly from 98% to 2%; from 13.1 to 15 min, 98% phase A and 2% phase B are used for elution; The mass spectrometry conditions are as follows: the capillary voltage of the ion source is set to 2500 V in the positive ion mode and -2000 V in the negative ion mode; the cone voltage is set to 30 V; the ion source temperature is 100 °C; the desolvation gas temperature is 500 °C; the backflush gas flow rate is 50 L / h; the desolvation gas flow rate is 800 L / h; the mass-to-charge ratio acquisition range is 50 - 1200; The relative expression levels of metabolites are obtained from the peak chromatograms obtained by liquid chromatography-mass spectrometry.

2. The method for identifying three varieties of medicinal Polygonatum sibiricum as claimed in claim 1, wherein: The relative expression levels of metabolites are obtained from the peak chromatograms obtained by liquid chromatography-mass spectrometry through the following method: the metabolite analysis uses the original data collected by MassLynx V4.2 software and performs data processing operations including peak extraction and peak alignment through Progenesis QI software. Identification is based on the online METLIN database and public databases in Progenesis QI software, and at the same time, theoretical fragment identification is performed with the deviation of precursor ion mass number within 10 ppm and the deviation of fragment ion mass number within 20 ppm.

3. The method for identifying three varieties of medicinal Polygonatum sibiricum as claimed in claim 1 or 2, wherein: In step 1), an extraction solution containing an internal standard is used; the extraction solution containing an internal standard is prepared by adding L-2-chlorophenylalanine to a concentration of 20 mg / L in a methanol-acetonitrile aqueous solution.

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