Method for identifying polygonum viviparum rhizomes and adulterants thereof

Abstract

The invention belongs to the technical field of medicinal material identification, and particularly relates to a method for identifying polygonum viviparum rhizomes and adulterants thereof, and the method for identifying the polygonum viviparum rhizomes and adulterants thereof comprises the step of identifying the polygonum viviparum rhizomes and adulterants thereof by detecting the contents of rutin and isoquercitrin in a rhizome sample to be detected. According to the identification method established by the invention, the polygonum viviparum rhizomes and the four polygonum viviparum rhizome adulterants are identified according to the contents of rutin and isoquercitrin in the rhizome sample to be detected, and a scientific basis is provided for quality evaluation and improvement of the polygonum viviparum rhizomes.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicinal material identification, and particularly relates to a method for identifying Bistorta vivipara (L.) Gray rhizome and its confused products. BACKGROUND

[0002] Bistorta vivipara (L.) Gray is a perennial herb of the Polygonaceae Bistorta, which is an important feed for the host insect of Cordyceps sinensis and has significant application value. Bistorta vivipara (L.) Gray usually grows in the regions of 1200 m to 5100 m of mountain slope, forest, alpine or subalpine meadow, etc. The dried rhizome is a traditional Tibetan medicinal material.

[0003] Bistorta vivipara (L.) Gray is confused with Bistorta, Polygonum, Fagopyrum and Polygonum, which are close relatives of Bistorta. However, the medicinal parts of these medicinal materials are all rhizomes, and the external morphologies are extremely similar, which leads to confusion and misuse in the market, which not only affects the efficacy, but also may cause potential risks to the health of patients.

[0004] At present, in the field of identification of Bistorta vivipara (L.) Gray rhizome and its confused products, there is no other related report of identification method except polymerase chain reaction (PCR). However, the PCR identification technology has limitations such as long experimental period, which limits its wide application to some extent.

[0005] Therefore, the research on the identification method of Bistorta vivipara (L.) Gray rhizome and its confused products is beneficial to fill the gap in this field and improve the quality management level of Bistorta vivipara (L.) Gray rhizome. SUMMARY

[0006] The main purpose of the present application is to provide a method for identifying Bistorta vivipara (L.) Gray rhizome and its confused products. The identification method established by the present application identifies Bistorta vivipara (L.) Gray rhizome and four kinds of confused products of Bistorta vivipara (L.) Gray rhizome by detecting the content of rutin and isoquercitrin in the rhizome sample to be tested, and provides a scientific basis for the quality evaluation of Bistorta vivipara (L.) Gray rhizome.

[0007] The present application provides a method for identifying Bistorta vivipara (L.) Gray rhizome and its confused products, which comprises: identifying Bistorta vivipara (L.) Gray rhizome and its confused products by detecting the content of rutin and isoquercitrin in the rhizome sample to be tested.

[0008] In some embodiments, the identification method comprises: S1) preparation of a reference solution, the reference comprising rutin and isoquercitrin; S2) preparation of a test solution: the rhizome sample to be tested is extracted with an organic solvent to prepare a test solution; S3) identification analysis: the reference solution and the test solution are detected by high performance liquid chromatography, and Bistorta vivipara (L.) Gray rhizome and its confused products are identified by the content of rutin and isoquercitrin in the rhizome sample to be tested.

[0009] In some embodiments, the adulterants include rhizomes of other Polygonaceae plants and / or rhizomes of plants in the genus Bistorta.

[0010] In some embodiments, the adulterants include at least one of rhizomes of Fagopyrum cymosum, rhizomes of Bistorta, rhizomes of Polygonum amplexicaule, and rhizomes of Polygonum flaccidum.

[0011] In some embodiments, when the mass percentage of rutin in the rhizome sample is ≥ A%, and the mass percentage of isoquercitrin is ≥ B%, the rhizome sample is determined to be rhizomes of Polygonum viviparum; or when the mass percentage of rutin in the rhizome sample is ≥ A%, and the mass percentage of isoquercitrin is < B%, the rhizome sample is determined to be rhizomes of Fagopyrum cymosum; or when the mass percentage of rutin in the rhizome sample is < A%, and the mass percentage of isoquercitrin is < B%, the rhizome sample is determined to be any one of rhizomes of Bistorta, rhizomes of Polygonum amplexicaule, and rhizomes of Polygonum flaccidum, in terms of the dry weight of the rhizome sample; the A% is 0.0010% to 0.0020%; and the B% is 0.0010% to 0.0020%. In some embodiments, the A% can be one of 0.0010%, 0.0011%, 0.0012%, 0.0013%, 0.0014%, 0.0015%, 0.0016%, 0.0017%, 0.0018%, 0.0019%, 0.0020% or any value within the above range. In some embodiments, the B% can be one of 0.0010%, 0.0011%, 0.0012%, 0.0013%, 0.0014%, 0.0015%, 0.0016%, 0.0017%, 0.0018%, 0.0019%, 0.0020% or any value within the above range. In some embodiments, the A% is 0.0014% and the B% is 0.0016%.

[0012] In some embodiments, the method further comprises: (i) drying another portion of the rhizome sample at 55°C for 5 to 6 hours, and obtaining the dry weight percentage of the rhizome sample by deducting the mass percentage of weight loss; or (ii) drying the rhizome sample at 55°C for 5 to 6 hours before step S2. In some embodiments, the drying time of the rhizome sample at 55°C can be one of 5 hours, 5.1 hours, 5.2 hours, 5.3 hours, 5.4 hours, 5.5 hours, 5.6 hours, 5.7 hours, 5.8 hours, 5.9 hours, 6 hours or any value within the above range.

[0013] In some embodiments, the chromatographic conditions of the high performance liquid chromatography comprise: the chromatographic column of the high performance liquid chromatography is a C18 chromatographic column, the mobile phase A of the high performance liquid chromatography is 0.1% vol acetic acid aqueous solution, and the mobile phase B is acetonitrile.

[0014] In some embodiments, the chromatographic column is a Waters XBridge BEH C18, and the inner diameter of the chromatographic column is 2.1mm-4.6mm. In some embodiments, the inner diameter of the chromatographic column can be one of 2.1mm, 2.5mm, 3.0mm, 3.5mm, 4mm, 4.5mm, 4.6mm or any value meeting the above range. In some embodiments, the inner diameter of the chromatographic column is 4.6mm.

[0015] In some embodiments, the column length of the chromatographic column is 30mm-75mm. In some embodiments, the column length of the chromatographic column can be one of 30mm, 40mm, 50mm, 60mm, 70mm, 75mm or any value meeting the above range. In some embodiments, the column length of the chromatographic column is 50mm.

[0016] In some embodiments, the particle size of the chromatographic column is 2.7μm.

[0017] In some embodiments, the chromatographic column is a Waters XBridge BEH C18 chromatographic column with an inner diameter of 4.6mm, a column length of 50mm, and a particle size of 2.7μm.

[0018] In some embodiments, the mobile phase A is 0.1% vol acetic acid aqueous solution, and the mobile phase B is acetonitrile.

[0019] In some embodiments, the elution procedure of the high performance liquid chromatography is isocratic elution.

[0020] In some embodiments, the volume ratio of the mobile phase A to the mobile phase B during elution of the high performance liquid chromatography is 85:15-92:8. In some embodiments, the volume ratio of the mobile phase A to the mobile phase B during elution of the high performance liquid chromatography can be one of 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8 or any value meeting the above range. In some embodiments, the volume ratio of the mobile phase A to the mobile phase B during elution of the high performance liquid chromatography is 89:11. In some embodiments, the volume ratio of the mobile phase A to the mobile phase B during elution of the high performance liquid chromatography is 88:12.

[0021] In some embodiments, the elution flow rate of the high-performance liquid chromatography (HPLC) is 0.9 mL / min to 1.1 mL / min. In some embodiments, the elution flow rate of the HPLC can be one of 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, or any value satisfying the above range. In some embodiments, the elution flow rate of the HPLC is 1 mL / min.

[0022] In some embodiments, the column temperature of the high-performance liquid chromatography (HPLC) is 20°C to 30°C. In some embodiments, the column temperature of the HPLC can be one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C, or any value satisfying the above range. In some embodiments, the column temperature of the HPLC is 25°C.

[0023] In some embodiments, the detection wavelength is 330nm to 370nm. In some embodiments, the detection wavelength can be one of 330nm, 340nm, 350nm, 360nm, and 370nm, or any value satisfying the above range. In some embodiments, the detection wavelength is 350nm.

[0024] In some embodiments, during isocratic elution, the volume ratio of mobile phase A to mobile phase B is maintained at 88:12.

[0025] In some embodiments, during the preparation of the test solution, 20 mL to 60 mL of the organic solvent is added per 1 g of the rhizome sample to be tested. In some embodiments, the volume of the organic solvent added per 1 g of the rhizome sample to be tested can be one of 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 55 mL, or 60 mL, or any value satisfying the above range. In some embodiments, 40 mL of the organic solvent is added per 1 g of the rhizome sample to be tested.

[0026] In some embodiments, the organic solvent includes a methanol solution.

[0027] In some embodiments, the methanol solution used to prepare the test solution is a methanol-water solution with a volume percentage of 20% to 50%. In some embodiments, the methanol solution used to prepare the test solution can be a methanol-water solution with a volume percentage of 20%, 25%, 30%, 35%, 40%, 45%, or 50%, or any value satisfying the above range. In some embodiments, the methanol solution used to prepare the test solution is a methanol-water solution with a volume percentage of 30%.

[0028] In some embodiments, the preparation steps of the test solution include: weighing the root and stem sample powder to be tested, adding an organic solvent for ultrasonic extraction, filtering through a filter membrane, and taking the filtrate.

[0029] In some embodiments, the ultrasonic extraction time is 10 min to 60 min. In some embodiments, the ultrasonic extraction time can be one of 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min, or any value satisfying the above range. In some embodiments, the ultrasonic extraction time is 30 min.

[0030] In some embodiments, each 1 mL of the reference solution contains 0.001 mg to 0.02 mg of rutin and 0.001 mg to 0.02 mg of isoquercitrin. In some embodiments, the mass of rutin contained in each 1 mL of the reference solution may be one of 0.001 mg, 0.002 mg, 0.005 mg, 0.008 mg, 0.01 mg, 0.012 mg, 0.014 mg, 0.016 mg, 0.018 mg, or 0.02 mg, or any value satisfying the above range. In some embodiments, the mass of isoquercitrin contained in each 1 mL of the reference solution may be one of 0.001 mg, 0.002 mg, 0.005 mg, 0.008 mg, 0.01 mg, 0.012 mg, 0.014 mg, 0.016 mg, 0.018 mg, or 0.02 mg, or any value satisfying the above range. In some embodiments, each 1 mL of the reference solution contains 0.01 mg of rutin and 0.01 mg of isoquercitrin.

[0031] In some embodiments, the solvent used to prepare the reference standard mixed solution includes at least one of anhydrous methanol and methanol solution.

[0032] In some embodiments, the methanol solution used to prepare the reference standard mixed solution is a methanol-water solution with a volume percentage of 20% to 50%. In some embodiments, the volume percentage of the methanol solution used to prepare the reference standard mixed solution can be one of 20%, 22%, 25%, 28%, 30%, 32%, 34%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, or 50%, or any value satisfying the above range. In some embodiments, the methanol solution used to prepare the reference standard mixed solution is a 30% methanol-water solution.

[0033] In some embodiments, the preparation steps of the reference standard mixed solution include: taking rutin and isoquercitrin reference standards separately, and independently adding anhydrous methanol to prepare rutin reference standard stock solution and isoquercitrin reference standard stock solution respectively; accurately transferring the rutin reference standard stock solution and the isoquercitrin reference standard stock solution, and diluting and making up to volume with methanol solution to obtain the reference standard mixed solution.

[0034] In some embodiments, each 1 mL of the rutin reference standard stock solution contains 0.8 mg to 1.2 mg of rutin. In some embodiments, each 1 mL of the rutin reference standard stock solution contains 0.9 mg to 1.1 mg of rutin. In some embodiments, the mass of rutin contained in each 1 mL of the rutin reference standard stock solution can be one of 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, or 1.2 mg, or any value satisfying the above range.

[0035] In some embodiments, each 1 mL of the isoquercitrin reference standard stock solution contains 0.8 mg to 1.2 mg of isoquercitrin. In some embodiments, each 1 mL of the isoquercitrin reference standard stock solution contains 0.9 mg to 1.1 mg of isoquercitrin. In some embodiments, the mass of isoquercitrin contained in each 1 mL of the isoquercitrin reference standard stock solution can be one of 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, or 1.2 mg, or any value satisfying the above range.

[0036] Beneficial effects

[0037] One of the technical solutions of the present invention includes at least one of the following beneficial technical effects:

[0038] (1) This invention establishes a method for identifying Polygonum bulbiferum rhizomes and their adulterants. The method identifies Polygonum bulbiferum rhizomes and four adulterants by measuring the content of rutin and isoquercitrin in the rhizome samples to be tested, providing a scientific basis for improving the quality evaluation of Polygonum bulbiferum rhizomes.

[0039] (2) The identification method provided by the present invention can further identify the rhizomes of *Fagopyrum cymosum* and the rhizomes of the other three species of *Polygonum bulbiferum* by the content of rutin and isoquercitrin.

[0040] (3) The identification method provided by the present invention is simple to operate, low in cost, high in efficiency, high in accuracy, safe and environmentally friendly, and can distinguish a variety of mixed products.

[0041] Terminology Explanation

[0042] In the description of this invention, the term "multiple" refers to two or more unless otherwise expressly specified. Similarly, "multiple sets" refers to two or more sets unless otherwise expressly specified. "Multiple pieces" refers to two or more pieces unless otherwise expressly specified.

[0043] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0044] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0045] In the description of this invention, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent three cases: A exists, A and B exist simultaneously, and B exists. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship.

[0046] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0047] The terms “volume%”, “%vol”, and “%(v / v)” all refer to volume percentage.

[0048] The term "wt%" indicates a percentage by mass.

[0049] "The dry weight of the rhizome sample to be tested" means the weight of the rhizome sample to be tested after being sliced ​​or crushed and dried at 50℃-60℃ (e.g., 55℃) for 5-6 hours, or the dry weight of another rhizome sample to be tested after being sliced ​​or crushed and dried at 50℃-60℃ (e.g., 55℃) for 5-6 hours, after deducting the percentage of weight loss.

[0050] In this invention, the rutin is a compound with CAS number 153-18-4.

[0051] In this invention, isoquercitrin is a compound with CAS number 482-35-9.

[0052] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0053] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. In the drawings:

[0054] Figure 1 This is a high-performance liquid chromatography (HPLC) chromatogram of a mixed solution of rutin and isoquercitrin reference standards in an embodiment of the present invention.

[0055] Figure 2 This is an HPLC chromatogram of the test solution of Polygonum bulbiferum rhizome in an embodiment of the present invention.

[0056] Figure 3 This is an HPLC chromatogram of the test solution of the rhizome of *Polygonum bistorta* in an embodiment of the present invention.

[0057] Figure 4 This is an HPLC chromatogram of the buckwheat rhizome test solution in an embodiment of the present invention.

[0058] Figure 5 This is an HPLC chromatogram of the Polygonum cuspidatum rhizome test solution in an embodiment of the present invention.

[0059] Figure 6 This is an HPLC chromatogram of the test solution of Polygonum aviculare rhizome in an embodiment of the present invention. Detailed Implementation

[0060] Exemplary embodiments of the present invention will now be described in more detail with reference to specific examples. It should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0061] Unless otherwise specified, the experimental reagents used in the following examples are all conventional biochemical reagents; the raw materials, instruments, and equipment used in the following examples can all be obtained through commercial purchase or by existing methods; the amounts of experimental reagents used are all amounts used in conventional experimental operations; and the experimental methods used are all conventional methods unless otherwise specified. It should be further noted that the following descriptions are merely exemplary and not intended to limit the specific scope of the invention.

[0062] Reagent preparation

[0063] 1) 30% (v / v) methanol aqueous solution: Measure 30 mL of anhydrous methanol, add ultrapure water to 100 mL, and mix well.

[0064] 2) 0.1% (v / v) acetic acid aqueous solution: Transfer 1 mL of acetic acid to 1 L of ultrapure water, mix well, and sonicate for 15 min.

[0065] Before preparing the test solution, the following Polygonum bulbils and their adulterants were sliced ​​or crushed, then dried at 55°C for 5-6 hours and crushed into powder.

[0066] Example 1

[0067] The detection limit of the identification method for Polygonum bulbiferum rhizome and its adulterants was investigated. The experimental procedure is as follows:

[0068] (1) Solution preparation

[0069] Weigh 4.72 mg of rutin (purity 95.6%), place it in a 5 mL volumetric flask, add anhydrous methanol to dissolve and dilute to volume, shake well to prepare rutin reference standard stock solution.

[0070] Weigh 10.14 mg of isoquercitrin (purity 98.0%), place it in a 10 mL volumetric flask, add anhydrous methanol to dissolve and dilute to volume, shake well to prepare the isoquercitrin reference standard stock solution.

[0071] Accurately transfer 1 mL each of rutin and isoquercitrin reference standard stock solution into a 100 mL volumetric flask, dilute with 30% (v / v) methanol aqueous solution and make up to volume, shake well to obtain the mixed stock solution of reference standards.

[0072] Accurately transfer 1 mL of the mixed mother liquor of the reference standard into a 25 mL volumetric flask, add 30% (v / v) methanol aqueous solution to dilute and make up to volume, shake well to obtain the diluted mixed reference standard solution.

[0073] (2) Chromatographic conditions

[0074] The chromatographic column was a Welch Boltimate LP-C18 (4.6 mm × 50 mm × 2.7 μm). Isocratic elution was performed with 0.1% (v / v) acetic acid aqueous solution: acetonitrile = 88:12. The flow rate was 1 mL / min, the column temperature was 25 °C, the injection volume was 5 μL, and the detection wavelength was 350 nm.

[0075] Experimental Results: The detection limits of this identification method were determined by the concentrations of rutin and isoquercitrin peaks with a signal-to-noise ratio (S / N) ≥ 3 in the mixed standard dilution. The detection limit for rutin in the established method was 0.361 μg / mL (calculated based on the extraction method of adding 40 mL of the organic solvent per 1 g of the tested rhizome sample, corresponding to a mass percentage of 0.0014% in the tested rhizome sample), and the detection limit for isoquercitrin was 0.397 μg / mL (calculated based on the extraction method of adding 40 mL of the organic solvent per 1 g of the tested rhizome sample, corresponding to a mass percentage of 0.0016% in the tested rhizome sample).

[0076] Example 2

[0077] The applicability of the identification method for Polygonum bulbiferum rhizomes and its adulterants was investigated. The experimental procedure is as follows:

[0078] (1) The solution preparation and chromatographic conditions are the same as in Example 1.

[0079] (2) Preparation of test solution: Weigh 0.15g of the rhizome sample powder to be tested and place it in a centrifuge tube. Add 6mL of 30% (v / v) methanol aqueous solution and place it in an ultrasonic cleaner for ultrasonic extraction for 30min. Filter through a 0.22μm filter membrane and take the filtrate for testing.

[0080] The measurement results in Example 2 are detailed in Table 1.

[0081] Table 1. Identification results of Polygonum bulbiferum rhizomes and their adulterants using the method established in this invention.

[0082]

[0083] Note: "Not detected" means that the rutin content is <0.0014% and the isoquercitrin content is <0.0016%.

[0084] At the same time, according to Figure 1 The HPLC chromatograms of the reference standard mixture shown indicate that the rutin peak is present at a retention time of 9.1 min, and the isoquercitrin peak is present at a retention time of 10.2 min. By comparison... Figures 2 to 6 The elution peaks at retention times of 9.1 min and 10.2 min in the HPLC chromatograms of the test sample solutions indicate that rutin and isoquercitrin peaks were detected in the HPLC chromatograms of Polygonum viviparum rhizomes; rutin peaks were detected in the HPLC chromatograms of Fagopyrum esculentum rhizomes, but isoquercitrin peaks were not detected; and rutin and isoquercitrin peaks were not detected in the HPLC chromatograms of Polygonum bistorta rhizomes, Polygonum clavatum rhizomes, and Polygonum tinctorium rhizomes.

[0085] Experimental conclusion: Rutin and isoquercitrin can be used as differentiating components between the rhizomes of Polygonum bulbiferum and those of Polygonum bistorta, Fagopyrum cymosum, Polygonum clavatum, and Polygonum spathulatum to distinguish the rhizomes of Polygonum bulbiferum and its adulterants.

[0086] The specific identification criteria in this invention are as follows:

[0087] (1) Based on the dry weight of the rhizome sample to be tested, when the mass percentage of rutin in the rhizome sample is greater than or equal to 0.0014% and the mass percentage of isoquercitrin is greater than or equal to 0.0016%, it is determined to be the rhizome of Polygonum bulbiferum.

[0088] (2) Based on the dry weight of the rhizome sample to be tested, when the mass percentage of rutin in the rhizome sample is greater than or equal to 0.0014% and the mass percentage of isoquercitrin is less than 0.0016%, it is determined to be the rhizome of buckwheat.

[0089] (3) Based on the dry weight of the rhizome sample to be tested, if the mass percentage of rutin in the rhizome sample to be tested is less than 0.0014% and the mass percentage of isoquercitrin is less than 0.0016%, it is determined that it may be any of the following species: Polygonum bistorta rhizome, Polygonum clavatum rhizome, or Polygonum spathulatum rhizome.

[0090] Example 3

[0091] The accuracy of identification methods for Polygonum bulbiferum rhizomes and its adulterants was investigated. The experimental procedure is as follows:

[0092] (1) Sample gene sequencing and species identification

[0093] Rhizomes of Polygonum villosa, Polygonum bistorta, Fagopyrum cymosum, Polygonum clavatum, and Polygonum spathulatum were collected as test samples. PCR amplification and sequencing were performed using the universal primers ITS2F / ITS3R for plant ITS regions. The sequenced sequences were compared with the GenBank database in the NCBI system.

[0094] (2) The identification method of Polygonum bulbiferum rhizome and its adulterants established in this invention was used to identify the rhizome samples to be tested.

[0095] The experimental results in Example 3 are detailed in Table 2.

[0096] Table 2 shows the gene sequencing identification results of the rhizome samples tested in Example 3.

[0097]

[0098]

[0099] As can be seen from Tables 1 and 2, the identification results obtained by the identification method of Polygonum bulbiferum rhizome and its adulterants established in this invention are consistent with the gene sequencing identification results.

[0100] The method for identifying Polygonum bulbiferum rhizomes and its adulterants established in this invention has high accuracy and can be used to distinguish Polygonum bulbiferum rhizomes from Polygonum bistorta rhizomes, Fagopyrum cymosum rhizomes, Polygonum clavatum rhizomes, and Polygonum spathulatum rhizomes.

[0101] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for identifying the rhizome of Polygonum bulbiferum and its adulterants, characterized in that, The identification method includes: identifying the rhizome of Polygonum viviparum and its confused products by detecting the contents of rutin and isoquercitrin in the sample of the rhizome to be tested.

2. The method for identifying Polygonum bulbiferum rhizomes and its adulterants as described in claim 1, characterized in that, The identification method includes: S1) Preparation of a reference mixture solution, where the reference substances include rutin and isoquercitrin; S2) Preparation of a test sample solution: preparing a test sample solution by extracting the sample of the rhizome to be tested with an organic solvent; S3) Identification and analysis: performing high performance liquid chromatography (HPLC) detection on the reference mixture solution and the test sample solution, and identifying the rhizome of Polygonum viviparum and its confused products by the contents of rutin and isoquercitrin in the sample of the rhizome to be tested; Optionally, the confused products include rhizomes of other Polygonaceae plants and / or rhizomes of Bistorta plants except Polygonum viviparum; Optionally, the confused products include at least one of the rhizomes of Fagopyrum dibotrys, Bistorta officinalis, Polygonum amplexicaule, and Polygonum sinomontanum.

3. The method for identifying Polygonum bulbiferum rhizomes and its adulterants as described in claim 2, characterized in that, Calculated based on the dry weight of the sample of the rhizome to be tested, when the mass percentage of rutin in the sample of the rhizome to be tested ≥ A%, and the mass percentage of isoquercitrin ≥ B%, it is determined as the rhizome of Polygonum viviparum; or, Calculated based on the dry weight of the sample of the rhizome to be tested, when the mass percentage of rutin in the sample of the rhizome to be tested ≥ A%, and the mass percentage of isoquercitrin < B%, it is determined as the rhizome of Fagopyrum dibotrys; or, Calculated based on the dry weight of the sample of the rhizome to be tested, when the mass percentage of rutin in the sample of the rhizome to be tested < A%, and the mass percentage of isoquercitrin < B%, it is determined as any one of the species of the rhizomes of Bistorta officinalis, Polygonum amplexicaule, and Polygonum sinomontanum; The A% is 0.0010% - 0.0020% or 0.0014%; The B% is 0.0010% - 0.0020% or 0.0016%; Optionally, the method further includes: (i) drying another portion of the sample of the rhizome to be tested at 55°C for 5 - 6 hours, deducting the mass percentage of weight loss, and obtaining the dry weight mass percentage of the sample of the rhizome to be tested; Or, (ii) drying the sample of the rhizome to be tested at 55°C for 5 - 6 hours before step S2.

4. The method for identifying the rhizome of Polygonum bulbiferum and its adulterants as described in any one of claims 2 to 3, characterized in that, The chromatographic conditions of the high performance liquid chromatography include: the chromatographic column for the high performance liquid chromatography method is a C18 chromatographic column, the mobile phase A for the high performance liquid chromatography method is 0.1% vol acetic acid aqueous solution, and the mobile phase B is acetonitrile.

5. The method for identifying Polygonum bulbiferum rhizomes and its adulterants as described in claim 4, characterized in that, The chromatographic column is Welch Boltimate LP-C18, The inner diameter of the chromatographic column is 2.1 mm - 4.6 mm or 4.6 mm; and / or, The column length of the chromatographic column is 30 mm - 75 mm or 50 mm; and / or, The particle size of the packing of the chromatographic column is 2.7 μm; and / or, The chromatographic column is a Welch Boltimate LP-C18 chromatographic column with an inner diameter of 4.6 mm, a column length of 50 mm, and a particle size of the packing of 2.7 μm; and / or, The mobile phase A is 0.1% vol acetic acid aqueous solution, and the mobile phase B is acetonitrile; and / or, The elution program of the high performance liquid chromatography method is isocratic elution; During the elution process of the high performance liquid chromatography method, the volume ratio of the mobile phase A to the mobile phase B is 85:15 - 92:8, or 89:11 or 88:12; and / or, The elution flow rate of the high-performance liquid chromatography method is 0.9 mL / min to 1.1 mL / min or 1 mL / min; and / or, The column temperature for the high-performance liquid chromatography method is 20℃~30℃ or 25℃; and / or, The detection wavelength is 330nm to 370nm or 350nm.

6. The method for identifying Polygonum bulbiferum rhizomes and its adulterants as described in claim 5, characterized in that, During the isocratic elution, the volume ratio of mobile phase A to mobile phase B is maintained at 88:

12.

7. The method for identifying the rhizome of Polygonum bulbiferum and its adulterants as described in any one of claims 2 to 6, characterized in that, In the preparation of the test solution, 20 mL to 60 mL of the organic solvent is added for every 1 g of the rhizome sample to be tested. Optionally, 40 mL of the organic solvent is added for every 1 g of the rhizome sample to be tested; Optionally, the organic solvent includes a methanol solution; Optionally, the methanol solution used in preparing the test solution is a methanol aqueous solution with a volume percentage of 20% to 50%, preferably a methanol aqueous solution with a volume percentage of 30%.

8. The method for identifying the rhizome of Polygonum bulbiferum and its adulterants as described in any one of claims 2 to 7, characterized in that, The preparation steps of the test solution include: Weigh the powdered rhizome sample to be tested, add an organic solvent for ultrasonic extraction, filter through a filter membrane, and collect the filtrate.

9. The method for identifying Polygonum bulbiferum rhizomes and its adulterants as described in claim 8, characterized in that, The ultrasonic extraction time is 10 min to 60 min, preferably 30 min.

10. The method for identifying the rhizome of Polygonum bulbiferum and its adulterants as described in any one of claims 2 to 9, characterized in that, Each 1 mL of the aforementioned reference standard mixture contains 0.001 mg to 0.02 mg of rutin and 0.001 mg to 0.02 mg of isoquercitrin; Optionally, each 1 mL of the aforementioned reference solution contains 0.01 mg of rutin and 0.01 mg of isoquercitrin; Optionally, the solvent used to prepare the reference standard mixed solution includes at least one of anhydrous methanol and methanol solution; Optionally, the methanol solution used to prepare the reference standard mixed solution is a methanol aqueous solution with a volume percentage of 20% to 50%, preferably a 30% methanol aqueous solution; Optionally, the preparation steps of the reference standard mixed solution include: Take rutin and isoquercitrin reference standards separately, and add anhydrous methanol to each to prepare rutin reference standard stock solution and isoquercitrin reference standard stock solution, respectively; Accurately transfer the rutin reference standard stock solution and the isoquercitrin reference standard stock solution, and dilute and bring to volume with methanol solution to prepare the reference standard mixed solution; Optionally, each 1 mL of the rutin reference standard stock solution contains 0.8 mg to 1.2 mg or 0.9 mg to 1.1 mg of rutin; Optionally, each 1 mL of the isoquercitrin reference standard stock solution contains 0.8 mg to 1.2 mg or 0.9 mg to 1.1 mg of isoquercitrin.

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