Method for identifying polygonum viviparum rhizomes and adulterants

Abstract

The invention provides a method for identifying polygonum viviparum rhizomes and adulterants. The identification method comprises the following steps: preparing a reference substance solution containing rutin; extracting and purifying the rhizome sample to be detected to obtain a solution to be detected; under the condition of the same wavelength, respectively measuring the absorbance of the reference substance solution and the to-be-measured solution; and based on the absorbance of the reference substance solution and the solution to be detected, determining that the rhizome sample to be detected is the polygonum viviparum rhizome or the adulterant. The identification method can be used for accurately identifying the polygonum viviparum rhizomes and adulterants, is simple, efficient, low in cost, safe and environment-friendly, can realize high-throughput analysis, provides a scientific basis for quality evaluation of the polygonum viviparum rhizomes, and has important significance for guaranteeing the controllability of medicinal material quality and promoting the sustainable development of the cordyceps sinensis ecological industry.

Description

Technical Field

[0001] This application relates to the field of identification technology of traditional Chinese medicine, specifically to a method for identifying the rhizome of Polygonum bulbiferum from adulterants. Background Technology

[0003] Polygonum villosa, along with other plants in the genus Polygonum and its close relatives such as Polygonum hydropiper, Fagopyrum cymosum, Polygonum clavatum, and Polygonum spathulatum, holds significant value in traditional Chinese medicine. However, because the medicinal parts of these herbs are all rhizomes, their appearance is extremely similar, leading to confusion and misuse in the market. This not only affects their efficacy but may also pose potential risks to patients' health.

[0004] Currently, in the field of identification of Polygonum bulbiferum rhizomes and its adulterants, there are no reports on identification methods other than polymerase chain reaction (PCR). However, PCR identification technology has limitations such as long experimental cycles, which to some extent restricts its widespread application. Therefore, conducting research on identification methods for Polygonum bulbiferum rhizomes and its adulterants will help fill the gap in this field and improve the quality management level of Polygonum bulbiferum rhizomes. Summary of the Invention

[0005] This application aims to at least partially address the technical problems existing in the prior art. To this end, this application proposes a method for distinguishing the rhizomes of *Polygonum viviparum* from adulterants. This method can accurately distinguish the rhizomes of *Polygonum viviparum* from adulterants, and it is simple, efficient, and capable of high-throughput analysis. It provides a scientific basis for the quality evaluation of *Polygonum viviparum* rhizomes, and is of great significance for ensuring the controllability of medicinal material quality and promoting the sustainable development of the *Cordyceps sinensis* ecological industry.

[0006] In one aspect of this application, a method for identifying Polygonum villosa rhizomes from adulterants is provided. According to an embodiment of this application, the method includes: step S1: preparing a rutin reference solution; step S2: extracting and purifying the rhizome sample to be tested to obtain a test solution; step S3: measuring the absorbance of the reference solution and the test solution under the same wavelength conditions; step S4: determining, based on the absorbance of the reference solution and the test solution, whether the rhizome sample to be tested is Polygonum villosa rhizome or an adulterant.

[0007] The core of this application lies in utilizing the significantly different components between the rhizomes of *Polygonum viviparum* and its adulterants to achieve accurate identification of *Polygonum viviparum* rhizomes and adulterants. These significantly different components can be detected based on differences in absorbance. Furthermore, rutin is a common and inexpensive reference standard. The inventors attempted to use it as a comparative reference for identifying *Polygonum viviparum* rhizomes and adulterants, discovering that accurate identification of *Polygonum viviparum* rhizomes and adulterants can be achieved based on the difference in absorbance between the test solution and the rutin reference solution. Therefore, the method of this application only requires simple absorbance measurement to quickly obtain identification results. It is simple to operate, efficient, and enables high-throughput analysis, providing a scientific basis for the quality evaluation of *Polygonum viviparum* rhizomes. This is of great significance for ensuring the controllability of medicinal material quality and promoting the sustainable development of the *Cordyceps sinensis* ecological industry.

[0008] According to embodiments of this application, the method for distinguishing Polygonum bulbiferum rhizomes from adulterants may also have the following additional technical features: According to embodiments of this application, the adulterants include rhizomes of Polygonaceae plants other than Polygonum bulbiferum and / or rhizomes of Polygonum genus plants.

[0009] According to embodiments of this application, the adulterant includes at least one of the following: Polygonum bistorta rhizome, Fagopyrum cymosum rhizome, Polygonum hydropiper rhizome, and Polygonum spathulatum rhizome.

[0010] According to an embodiment of this application, the extraction process includes: ultrasonically extracting the powder of the rhizome sample to be tested with a first methanol aqueous solution, centrifuging the obtained extract, collecting the supernatant and mixing it with water to obtain an extraction product solution.

[0011] According to embodiments of this application, in the ultrasonic extraction, the powder of the rhizome sample to be tested, calculated on a dry weight basis, is supplemented with 4 ml to 7.5 ml of a first methanol aqueous solution for every 1 g of the powder. According to embodiments of this application, 4 ml, 4.5 ml, 5 ml, 5.5 ml, 6 ml, 6.5 ml, 7 ml, or 7.5 ml of the first methanol aqueous solution are added for every 1 g of the powder. This effectively extracts the differential components from the rhizomes of *Polygonum viviparum* and its adulterants, thereby improving the accuracy of subsequent detection.

[0012] It should be noted that the powder of the root and stem sample to be tested is calculated based on dry weight. When the root and stem sample to be tested is a solid powder, its weight is directly weighed and recorded as the dry weight (referred to as "dry weight"). When the root and stem sample to be tested is a non-solid powder, it can be obtained by slicing or crushing it, drying it, crushing it into a solid powder, and then weighing it. Specifically, the drying temperature can be 50-60℃, and the time can be 5-6 hours.

[0013] According to an embodiment of this application, before step S2, the rhizome sample to be tested is sliced ​​or crushed and then dried at 50-60°C for 5-6 hours to obtain the dried rhizome sample to be tested.

[0014] According to embodiments of this application, the methanol concentration in the first methanol aqueous solution is 40% to 80% by volume. For example, the methanol concentration in the first methanol aqueous solution is 50%, 55%, 60%, 65%, 70%, or 75%, preferably 50% by volume. This allows for the effective extraction of differential components from the rhizomes of Polygonum viviparum and its adulterants.

[0015] According to embodiments of this application, the volume ratio of the supernatant to water is 1:3 to 1:7.5, preferably 1:4. Exemplarily, the volume ratios of the supernatant to water are 1:3, 1:4, 1:5, 1:6, 1:7, and 1:7.5. This allows for the effective adsorption of differential components from the rhizomes of Polygonum bulbiferum and its adulterants onto the solid-phase extraction column.

[0016] According to embodiments of this application, the ultrasonic extraction time is 20 min-60 min. According to embodiments of this application, the ultrasonic extraction time is 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min. Therefore, differential components in the rhizomes of *Polygonum viviparum* and its adulterants can be effectively extracted.

[0017] According to an embodiment of this application, the purification process includes: adding the extracted product solution obtained from the extraction process into a solid-phase extraction column, then adding an impurity eluent to the solid-phase extraction column for impurity elution, and discarding the eluent; adding the target analyte eluent to the solid-phase extraction column, collecting the eluent, and obtaining the test solution.

[0018] Solid-phase extraction (SPE) columns, employing a selective adsorption-elution mechanism, can effectively separate differentially expressed components from impurities such as pigments and polysaccharides in a short time, efficiently enriching the target composition—the differentially expressed components. This ensures that subsequent absorbance detection is free from impurity interference, improving the accuracy of identification. Specifically, when the extracted product solution enters the SPE column and reaches the surface of the stationary phase (ODS), the target differentially expressed components and some impurities in the solution interact with and are adsorbed onto the ODS stationary phase. At this point, a first eluent is added to elute the impurities, while the differentially expressed components remain bound to the stationary phase. Next, a target eluent is added to disrupt the binding force between the differentially expressed components and the stationary phase, eluting and collecting the differentially expressed components.

[0019] According to embodiments of this application, the solid-phase extraction column packing material is octadecylsilane-bonded silica gel. This allows for the effective separation of differentially expressed components from impurities such as pigments and polysaccharides in a short time, improving the accuracy of identification.

[0020] According to an embodiment of this application, the impurity eluent is selected from a second methanol aqueous solution, wherein the methanol concentration in the second methanol aqueous solution is 10% to 30% by volume. Exemplarily, the methanol concentration in the second methanol aqueous solution is 10%, 15%, 20%, 25%, or 30%, preferably 20% by volume. Thus, impurities are effectively removed without damaging the binding force between the differential components and the solid-phase extraction column, ensuring that subsequent absorbance detection is free from impurity interference and improving the accuracy of identification.

[0021] According to embodiments of this application, the target analyte eluent is selected from a third methanol aqueous solution, wherein the methanol concentration in the third methanol aqueous solution is 50% to 100% by volume. Exemplarily, the methanol concentration in the third methanol aqueous solution is 50%, 60%, 70%, 80%, 90%, or 100% by volume, preferably 60% by volume. This effectively disrupts the binding force between the differentially expressed components and the solid-phase extraction column, thereby achieving the elution of significantly differentially expressed components.

[0022] According to embodiments of this application, the volume ratio of the solid-phase extraction column to the eluent is 1:(4~8), exemplarily 1:4, 1:5, 1:6, 1:7.4, 1:8, preferably 1:7.4. Thus, the eluent can effectively elute impurities in the extract solution from the stationary phase, achieving the purpose of impurity removal.

[0023] According to embodiments of this application, the volume ratio of the solid-phase extraction column to the target eluent is 1:(4~8), exemplarily 1:4, 1:5, 1:6, 1:7.4, 1:8, preferably 1:7.4. Thus, the target eluent can sufficiently disrupt the binding force between the differential component and the stationary phase, eluting and collecting the differential component for identifying Polygonum bulbiferum rhizomes from adulterants.

[0024] According to embodiments of this application, the wavelength is 350 nm to 380 nm. Exemplarily, the wavelengths are 350 nm, 360 nm, 370 nm, and 380 nm, preferably 360 nm. At this wavelength, the differential components can effectively distinguish the rhizomes of Polygonum bulbiferum from adulterants.

[0025] According to an embodiment of this application, the method further includes detecting the absorbance of the third methanol aqueous solution and the absorbance of the solvent in the reference solution under the same wavelength conditions as in step S3.

[0026] According to embodiments of this application, based on the absorbance of the reference solution and the test solution, the rhizome sample to be tested is determined to be either *Polygonum bulbiferum* rhizome or a mixed sample according to the following criteria: Criterion: If the absorbance of the test solution is greater than the absorbance of the reference solution, the rhizome sample is determined to be *Polygonum bulbiferum* rhizome; if the absorbance of the test solution is not greater than the absorbance of the reference solution, the rhizome sample is determined to be a mixed sample. Wherein, the third methanol aqueous solution in the test solution may be the same as or different from the solvent in the reference solution, the absorbance of the test solution is the absorbance obtained after deducting the absorbance of the third methanol aqueous solution in the test solution, and the absorbance of the reference solution is the absorbance obtained after deducting the absorbance of the solvent in the reference solution. Alternatively, the third methanol aqueous solution in the test solution is the same solvent as the reference solution, the absorbance of the test solution is the absorbance before deducting the absorbance of the third methanol aqueous solution in the test solution, and the absorbance of the reference solution is the absorbance before deducting the absorbance of the solvent in the reference solution.

[0027] Since the main components in the test sample solutions of *Polygonum viviparum* rhizomes and adulterants are different, the absorbance of the test sample solutions will show certain differences. By comparing the absorbance of a reference solution of a specific concentration with the test solution and using the above-mentioned judgment method, *Polygonum viviparum* rhizomes and adulterants can be accurately identified.

[0028] According to embodiments of this application, the concentration of rutin in the reference solution is 0.045 mg / mL to 0.055 mg / mL, exemplarily 0.045 mg / mL, 0.046 mg / mL, 0.048 mg / mL, 0.050 mg / mL, 0.052 mg / mL, 0.054 mg / mL, 0.055 mg / mL, preferably 0.050 mg / mL.

[0029] According to an embodiment of this application, when the concentration of the rutin reference solution is not between 0.045 mg / mL and 0.055 mg / mL, before making the determination, the absorbance measured in the reference solution is converted to the absorbance corresponding to a rutin concentration of 0.045 mg / mL to 0.055 mg / mL (for example, 0.045 mg / mL, 0.046 mg / mL, 0.048 mg / mL, 0.050 mg / mL, 0.052 mg / mL, 0.054 mg / mL, or 0.055 mg / mL), preferably converted to the absorbance corresponding to 0.050 mg / mL, and recorded as the converted absorbance. The converted absorbance and the absorbance of the test solution are then compared according to the determination criteria. This improves the accuracy of identification.

[0030] According to an embodiment of this application, the reference solution contains rutin, methanol, and water.

[0031] According to the embodiments of this application, the reference solution is prepared using a 50% to 75% volume-volume methanol aqueous solution as a solvent, preferably a 60% volume-volume methanol aqueous solution as a solvent.

[0032] According to the embodiments of this application, the concentration of rutin in the reference solution is 0.045 mg / mL to 0.055 mg / mL.

[0033] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application.

[0034] Beneficial effects Compared with the prior art, the present invention has at least one of the following beneficial effects: (1) The method for identifying Polygonum bulbil rhizomes and its adulterants provided by the present invention has the advantages of simple operation, safety and environmental protection, high accuracy, low cost and high efficiency, and can realize high-throughput analysis.

[0035] (2) The method for identifying Polygonum bulbiferum rhizomes and its adulterants provided by the present invention does not require expensive detection equipment (such as high performance liquid chromatography, nuclear magnetic resonance or mass spectrometry), expensive reagents and chromatographic columns, or gene sequencing, which greatly reduces the cost required for identification. Detailed Implementation

[0036] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0037] The terms “volume%” and “% (v / v)” represent volume percentages.

[0038] The term "wt%" indicates a percentage by mass.

[0039] The term "dry product" refers to the product obtained by drying the sample slices or pulverizes at 50-60°C for 5-6 hours.

[0040] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0041] 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.

[0042] 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.

[0043] "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 the 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.

[0044] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0045] In the following examples, the test samples (Polygonum bulbil rhizomes and adulterants) were sliced ​​or pulverized and dried at 55°C for 5-6 hours before the test solution was prepared.

[0046] Column volume: The sum of the void volume between the packing particles and the volume of the packing particles in a solid phase extraction column, which is the product of the cross-sectional area corresponding to the inner diameter of the solid phase extraction column and the column height occupied by the packing.

[0047] Example 1 In this embodiment, the rhizomes of Polygonum bulbiferum and adulterants (Polygonum hydropiper rhizomes, Fagopyrum cymosum rhizomes, Polygonum clavatum rhizomes, or Polygonum spathulatum rhizomes) are identified according to the following methods: Step 1: Preparation of reference solution: Weigh an appropriate amount of rutin reference standard, add 60% (v / v) methanol aqueous solution to dissolve it, and prepare a reference solution with a concentration of approximately 0.05 mg / mL.

[0048] Step 2: Preparation of the test solution to be purified: Take the sample and crush it. Take 2 g of the powder and place it in a centrifuge tube. Add 10 mL of 50% (v / v) methanol aqueous solution and place it in an ultrasonic cleaner for ultrasonic extraction for 30 min. Take it out and centrifuge for 5 min. Take 1 mL of the supernatant and mix it with 4 mL of ultrapure water to obtain the test solution to be purified.

[0049] The samples included the rhizomes of Polygonum bulbiferum, Polygonum hydropiper, Fagopyrum cymosum, Polygonum cuspidatum, and Polygonum tinctorium, which were tested separately.

[0050] Step 3: Preparation of solid-phase extraction column: The column (inner diameter 0.83 cm) was packed with methanol-activated octadecylsilane-bonded silica gel (ODS) to a height of 1 cm and a volume of 0.54 mL. The top was covered with filter paper of the same cross-sectional size, and the methanol solution inside the column was replaced with ultrapure water.

[0051] Step 4, Purification: Add 2 mL of the test sample solution to be purified onto the solid phase extraction column. When the solution reaches the stationary phase (ODS) section, add 4 mL of 20% (v / v) methanol aqueous solution as the impurity elution solvent to elute the impurities. Discard the eluent. Then add 4 mL of 60% (v / v) methanol aqueous solution as the target analyte elution solvent to elute the target analyte. Collect the eluent to obtain the test sample solution.

[0052] Step 5, Sample determination: Take 200 μL each of the reference solution and the test solution into a quartz 96-well plate, and measure the absorbance at 360 nm using an ELISA reader.

[0053] Step 6: Judgment: Compare the absorbance of the test solution with the absorbance of the 0.05 mg / mL rutin reference solution. When the absorbance of the test solution is greater than that of the 0.05 mg / mL rutin reference solution, the test sample is determined to be the rhizome of Polygonum viviparum; when the absorbance of the test solution is less than or equal to that of the 0.05 mg / mL rutin reference solution, the test sample is determined to be the rhizome of Polygonum bistorta, Fagopyrum cymosum, Polygonum clavatum, or Polygonum spathulatum.

[0054] The results are shown in Table 1. It can be seen that the method of this application can effectively and accurately identify the rhizomes of Polygonum villosa, Polygonum hydropiper, Fagopyrum cymosum, Polygonum clavatum, and Polygonum spp.

[0055] Table 1 Sample Measurement Results

[0056] Note: Each type of rhizome has a different number, indicating its different origin.

[0057] Example 2 Sample gene sequencing and species identification Rhizomes of Polygonum villosa, Polygonum bistorta, Fagopyrum cymosum, Polygonum clavatum, and Polygonum spathulatum from different sources were collected and subjected to PCR amplification using the universal primers ITS2F / ITS3R for plant ITS regions. The sequenced sequences were then compared with the GenBank database in the NCBI system.

[0058] Experimental results: The identification results of the method established in this application are consistent with the gene sequencing identification results (Tables 1 and 2). The established method has high accuracy and can be used to identify the rhizomes of Polygonum bulbiferum from adulterants (Polygonum hydropiper rhizomes, Fagopyrum cymosum rhizomes, Polygonum clavatum rhizomes, and Polygonum spathulatum rhizomes).

[0059] Table 2. Sample gene sequencing identification results

[0060] Example 3 Ultrasonic extraction was performed according to the method in step 2 of Example 1, except that the concentration of methanol in the methanol-water solution was replaced with 25% (v / v), 75% (v / v), or 100% (v / v), or ultrapure water was replaced with methanol-water solution. 0.5 mL of the supernatant from step 2 was mixed with 9.5 mL of 60% (v / v) methanol-water solution to obtain the total extract test solution. 200 μL of each total extract test solution was added to a 96-well quartz plate, and the absorbance was measured at 360 nm using a microplate reader.

[0061] The absorbance of the total extract test solution prepared using Polygonum bulbil rhizome as sample is shown in Table 3. It can be seen that the extraction rate of compounds in the sample is higher when using 50% (v / v) and 75% (v / v) methanol aqueous solution.

[0062] Table 3. Investigation of extraction solvents

[0063] Example 4 The method of Example 1 was used to identify Polygonum villosa rhizomes and adulterants, except that in step 4, the concentration of methanol in the impurity elution solvent was adjusted to 10% (v / v), 30% (v / v), and 40% (v / v), respectively.

[0064] The absorbance of the test solutions prepared using Polygonum bulbiferum rhizomes and adulterants as samples are shown in Table 4.

[0065] When the ratio of impurity elution solvent is between 10% (v / v) and 30% (v / v) methanol aqueous solution, the identification effect can be achieved.

[0066] When the proportion of impurity elution solvent is ≥40% (v / v) methanol aqueous solution, it is impossible to effectively distinguish between the rhizomes of Polygonum bulbiferum and Polygonum hydropiper rhizomes, that is, it is impossible to distinguish between the rhizomes of Polygonum bulbiferum and adulterants.

[0067] Table 4 Investigation of impurity elution solvents

[0068] Example 5 The method of Example 1 was used to identify Polygonum villosa rhizomes and adulterants, except that in step 4, the concentration of methanol in the elution solvent of the target analyte was adjusted to 40% (v / v), 50% (v / v), and 100% (v / v), respectively.

[0069] The absorbance of the test solutions prepared using Polygonum bulbiferum rhizomes and adulterants as samples are shown in Table 5. It can be seen that when the elution solvent ratio of the target analyte is 50% (v / v)-100% (v / v) methanol aqueous solution, the identification of Polygonum bulbiferum rhizomes and adulterants can be achieved.

[0070] When the elution solvent ratio of the target substance is ≤40% (v / v) methanol aqueous solution, it is not possible to effectively distinguish between the rhizomes of Polygonum bulbiferum and the rhizomes of Polygonum hydropiper, that is, it is not possible to distinguish between the rhizomes of Polygonum bulbiferum and adulterants.

[0071] Table 5. Investigation of elution solvents for target analytes

[0072] Example 6 The method of Example 1 was used to identify the rhizomes of Polygonum bulbiferum and adulterants, except that in step 5, the detection wavelengths were adjusted to 340 nm, 350 nm, 370 nm, 380 nm and 390 nm, respectively.

[0073] The absorbance of the test solutions prepared using Polygonum villosa rhizomes and adulterants as samples is shown in Table 6. It can be seen that the detection wavelengths of 350 nm to 380 nm can achieve the identification effect.

[0074] When the detection wavelength is 340 nm, it is impossible to distinguish between the rhizomes of Polygonum bulbiferum and Polygonum claspingii.

[0075] When the detection wavelength is 390 nm, it is impossible to distinguish between the rhizomes of Polygonum bulbiferum, Polygonum clavatum, and Polygonum wingedis.

[0076] Table 6. Investigation of Detection Wavelengths

[0077] Example 7 The method of Example 1 was used to identify the rhizomes of Polygonum bulbiferum and adulterants. The difference was that in step 6, the absorbance of the rutin reference solution after deducting the absorbance of the blank solvent (60% (v / v) methanol aqueous solution) was converted into absorbance values ​​of 0.035 mg / mL, 0.045 mg / mL, 0.050 mg / mL, 0.055 mg / mL, 0.060 mg / mL, and 0.065 mg / mL, respectively, based on the actual concentration and in a directly proportional linear relationship (y=kx, where x is the concentration, y is the absorbance, and k is the proportionality coefficient, which can be calculated by substituting the actual concentration and actual absorbance into y=kx).

[0078] The absorbance of the test solutions prepared using Polygonum villosa rhizomes and adulterants as samples, after subtracting the absorbance of the blank solvent (60% (v / v) methanol aqueous solution), is shown in Table 7. It can be seen that the absorbance of the rutin reference solution with a concentration of 0.045 mg / mL to 0.055 mg / mL and the absorbance of the test solution can both achieve the identification effect.

[0079] When converting the absorbance of the rutin reference solution to absorbances of 0.035 mg / mL and 0.065 mg / mL based on the actual concentration, there is a possibility of identification errors.

[0080] Table 7. Investigation of Rutin Conversion Concentration

[0081] Example 8 The method of Example 1 was used to identify the rhizomes of Polygonum bulbiferum and adulterants. The difference was that the impurity elution step in step 4 was not performed. Instead, when the solution was placed to the cross section of the stationary phase (ODS), 4 mL of 60% (v / v) methanol aqueous solution was directly added as the target analyte elution solvent to elute the target analyte. The eluent was collected to obtain the test solution.

[0082] The results showed that the purification of the test solution without the impurity elution step could not achieve the identification of Polygonum bulbiferum rhizomes and its adulterants.

[0083] Table 8 Comparison of steps without impurity elution

[0084] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A method for distinguishing the rhizomes of Polygonum bulbiferum from adulterants, characterized in that, include: Step S1: Prepare a reference solution of rutin; Step S2: Extract and purify the rhizome sample to be tested to obtain the test solution; Step S3: Under the same wavelength conditions, measure the absorbance of the reference solution and the test solution respectively; Step S4: Based on the absorbance of the reference solution and the test solution, determine whether the rhizome sample to be tested is Polygonum bulbil rhizome or a mixed product.

2. The method according to claim 1, characterized in that, The adulterants include the rhizomes of other Polygonaceae plants and / or the rhizomes of Bistortae plants, excluding Polygonum bulbiferum. Optionally, the adulterant includes at least one of the following: Polygonum hydropiper rhizome, Fagopyrum cymosum rhizome, Polygonum hydropiper rhizome, and Polygonum spathulatum rhizome.

3. The method according to claim 1, characterized in that, The extraction process includes: The powder of the rhizome sample to be tested was ultrasonically extracted with the first methanol aqueous solution. The resulting extract was then centrifuged, and the supernatant was collected and mixed with water to obtain the extract product solution. Optionally, in the ultrasonic extraction, the powder of the rhizome sample to be tested is calculated on a dry weight basis, and 4 ml to 7.5 ml of a first methanol aqueous solution is added for every 1 g of the powder of the rhizome sample to be tested.

4. The method according to claim 3, wherein the methanol concentration in the first methanol aqueous solution is 40% to 80% by volume, preferably 50% by volume. Optionally, the mixing volume ratio of the supernatant to water is 1:3 to 1:7.5, preferably 1:

4.

5. The method according to any one of claims 1-4, characterized in that, The purification process includes: The extracted product solution obtained from the extraction process is added to a solid phase extraction column, and then an impurity eluent is added to the solid phase extraction column to elute the impurities. The eluent is then discarded. The target analyte eluent is added to the solid-phase extraction column, and the eluent is collected to obtain the test solution. Optionally, the solid-phase extraction column packing material is octadecylsilane-bonded silica gel.

6. The method according to any one of claims 1-5, characterized in that, Before step S2, the rhizome sample to be tested is sliced ​​or crushed and then dried at 50-60℃ for 5-6 hours to obtain the dried rhizome sample to be tested.

7. The method according to any one of claims 5-6, characterized in that, The impurity eluent is selected from a second methanol aqueous solution, wherein the methanol concentration in the second methanol aqueous solution is 10% to 30% by volume, preferably 20% by volume. Optionally, the eluent for the target substance is selected from a third methanol aqueous solution, wherein the methanol concentration in the third methanol aqueous solution is 50% to 100% by volume, preferably 60% by volume. Optionally, the volume ratio of the solid-phase extraction column to the impurity eluent is 1:(4~8), preferably 1:7.4; Optionally, the volume ratio of the solid-phase extraction column to the target eluent is 1:(4~8), preferably 1:7.

4.

8. The method according to any one of claims 1-7, characterized in that, The wavelength is 350 nm to 380 nm, preferably 360 nm.

9. The method according to any one of claims 1-8, characterized in that, It also includes detecting the absorbance of the third methanol aqueous solution and the absorbance of the solvent in the reference solution under the same wavelength conditions as in step S3.

10. The method according to any one of claims 1-9, characterized in that, The reference solution is prepared using a 50% to 100% volume methanol aqueous solution as the solvent, preferably a 60% volume methanol aqueous solution as the solvent, and the rutin concentration in the reference solution is 0.045 mg / mL to 0.055 mg / mL, preferably 0.050 mg / mL. Based on the absorbance of the reference solution and the test solution, the rhizome sample to be tested is determined to be either Polygonum bulbiferum rhizome or a mixed product according to the following criteria: Judgment criteria: If the absorbance of the test solution is greater than that of the reference solution, the rhizome sample is determined to be a Polygonum bulbiferum rhizome; if the absorbance of the test solution is not greater than that of the reference solution, the rhizome sample is determined to be a mixed sample. Wherein, the third methanol aqueous solution in the test solution may be the same as or different from the solvent in the reference solution, the absorbance of the test solution is the absorbance obtained after deducting the absorbance of the third methanol aqueous solution in the test solution, and the absorbance of the reference solution is the absorbance obtained after deducting the absorbance of the solvent in the reference solution. Alternatively, the third methanol aqueous solution in the test solution is the same solvent as the reference solution, the absorbance of the test solution is the absorbance before deducting the absorbance of the third methanol aqueous solution in the test solution, and the absorbance of the reference solution is the absorbance before deducting the absorbance of the solvent in the reference solution.

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