A method for constructing HPLC characteristic spectrum of processed Polygonatum sibiricum

Abstract

The present invention provides a method for constructing an HPLC characteristic spectrum of a prepared polygonatum, comprising: A) dissolving a prepared polygonatum raw material in a solvent to obtain a liquid to be tested; B) determining the liquid to be tested by high performance liquid chromatography to obtain an HPLC characteristic spectrum of the prepared polygonatum raw material; the HPLC chromatographic conditions are as follows: a C18 column; mobile phase A is acetonitrile; mobile phase B is a 0.1% aqueous phosphoric acid solution, and gradient elution. The present invention adopts high performance liquid chromatography, selects acetonitrile-0.1% phosphoric acid as the mobile phase for gradient elution, and uses uridine, 5-hydroxymethylfurfural, and daidzein as reference substances to establish an HPLC characteristic spectrum of the prepared polygonatum. The method has good repeatability and precision, is stable, and is reliable, and can control the quality of the prepared polygonatum.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, in particular to a method for constructing an HPLC characteristic spectrum of processed polygonatum. Background Art

[0002] Polygonatum sibiricum is the dried rhizome of Polygonatum kingianum Coll. et Hemsl., Polygonatum sibiricum Red., or Polygonatum cyrtonema Hua, all of the Liliaceae family. It has a long history of medicinal use, documented in numerous historical compendiums of Materia Medica. It is currently listed in the 2020 edition of the Chinese Pharmacopoeia, Volume 1. It is sweet and neutral in nature. It enters the spleen, lung, and kidney meridians. It tonifies qi and nourishes yin, strengthens the spleen, moistens the lungs, and benefits the kidneys. It is used to treat spleen and stomach qi deficiency, fatigue, stomach yin deficiency, dry mouth and poor appetite, dry cough due to lung deficiency, hemoptysis caused by exertional cough, deficiency of essence and blood, soreness of the waist and knees, premature graying of hair, and internal heat and thirst. Processed Polygonatum sibiricum is a processed product of Polygonatum sibiricum. The preparation method involves taking black beans, decocting the resulting concentrated juice, and then boiling it with the Polygonatum sibiricum (the black bean juice should cover the surface of the herb). After boiling, simmer until the water is gone. Remove the herb, let it air dry, and then steam it in a container for 5-8 hours. Alternatively, soak the Polygonatum sibiricum in the black bean juice until it thoroughly moistens the core. Steam it until the inside and outside are moist and black. Remove the herb, cut it into thick slices, and dry it. For every 100kg of Polygonatum sibiricum, 10kg of black beans are used. (Sichuan Province Traditional Chinese Medicine Processing Specifications, 2015 edition). This processing enhances its spleen-tonifying and kidney-benefiting effects. Processed Polygonatum sibiricum has a diverse chemical composition, primarily including steroidal saponins, flavonoids, phenylpropanoids, alkaloids, and polysaccharides. Basic research on processed Polygonatum sibiricum slices, standard decoctions, extracts, and formulated granules is relatively weak both domestically and internationally. Standard control indicators are simplistic, making it difficult to effectively control the quality of the medicinal material. This has led to a long-standing phenomenon of varying quality in the slices and their finished pharmaceutical preparations. This study evaluated the quality of processed Polygonatum sibiricum slices, standard decoction, extract, granules and their preparations, and provided a basis for the identification of processed Polygonatum sibiricum and its related preparations.

[0003] Currently, there is little research on the quality of processed Polygonatum sibiricum slices, standard decoctions, extracts, formula granules, and their compound preparations, and there is a lack of an effective means for their quality control. The present invention aims to construct a high-performance liquid chromatography characteristic spectrum method for processed Polygonatum sibiricum to comprehensively reflect the quality level of processed Polygonatum sibiricum and its related preparations, and to provide guidance for the quality control of processed Polygonatum sibiricum slices, standard decoctions, extracts, formula granules, and their related preparations. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing an HPLC characteristic spectrum of processed Polygonatum sibiricum. The HPLC characteristic spectrum method of processed Polygonatum sibiricum constructed by the present invention is stable and reliable, and can control the quality of processed Polygonatum sibiricum.

[0005] The present invention discloses the construction and application of a high-performance liquid chromatography characteristic spectrum detection method for processed polygonatum. The method confirms 9 characteristic peaks in total, specifies their relative retention times, establishes a control characteristic spectrum, identifies 3 index components, uridine, 5-hydroxymethylfurfural, and daidzein, fully demonstrates the chemical composition characteristics of processed polygonatum, and comprehensively reflects the quality information of chemically processed polygonatum. Thus, the quality of processed polygonatum slices, standard decoctions, extracts, formula granules and related preparations can be comprehensively and effectively controlled.

[0006] This method can be used to identify Polygonatum sibiricum and its processed products. The chromatogram of the processed Polygonatum sibiricum sample shows nine characteristic peaks. Except for peaks 4, 6, 8, and 9, the retention times of all the peaks correspond to the retention times of the five characteristic peaks in the chromatogram of the Polygonatum sibiricum sample. Peak 4 (5-hydroxymethylfurfural), peak 6, peak 8 (daidzin), and peak 9 are the components of Polygonatum sibiricum after it is processed into processed Polygonatum sibiricum.

[0007] The method of the present invention can well distinguish processed polygonatum and polygonatum, and can be used for identifying processed polygonatum.

[0008] The present invention provides a method for constructing an HPLC characteristic spectrum of prepared Polygonatum sibiricum, comprising:

[0009] A) dissolving the raw material of Polygonatum odoratum by solvent to obtain a liquid to be measured;

[0010] B) liquid to be tested is adopted to high performance liquid chromatography to measure, and obtains the HPLC characteristic spectrum of polygonatum processed raw material;

[0011] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the elution is performed in a gradient manner.

[0012] The present invention provides a method for constructing an HPLC characteristic spectrum of prepared Polygonatum sibiricum. First, the prepared Polygonatum sibiricum raw material is taken and dissolved in a solvent to obtain a test solution. The solvent is preferably 30% methanol.

[0013] The present invention adopts the above extraction solvent, which has large chromatographic peak information and good effect.

[0014] Specifically, the raw materials for preparing polygonatum are dissolved in a solvent, extracted, cooled, shaken, and filtered to obtain the product.

[0015] The extraction method of the present invention is ultrasonic extraction or heating reflux extraction; preferably ultrasonic extraction; the ultrasonic power is preferably 600W, the frequency is preferably 40kHz; the extraction time is preferably 20 to 30 minutes; more preferably 20 minutes.

[0016] When the extraction time was 20 min, the chromatogram peak shape and separation were better.

[0017] Among them, the ratio of the mass g of the prepared Polygonatum odoratum raw material to the volume mL of the solvent is preferably (0.5-1): (10-50); more preferably (0.5-1): 10-20; and most preferably 0.5:10 for the decoction pieces, standard soups and formula granules.

[0018] The solvent is 30% methanol. When the extraction solvent is 30% methanol, the peak shapes of the characteristic peaks are good and the separation degree is moderate.

[0019] The raw materials for preparing polygonatum are prepared polygonatum slices, standard decoctions, extracts and formula granules. The present invention does not limit them, and the above raw materials can all be subjected to quality control and qualitative testing by the method of the present invention.

[0020] It also includes the preparation of reference substance solutions and the preparation of reference medicinal material solutions;

[0021] Preparation of reference substance solution: uridine, 5-hydroxymethylfurfural, and daidzein were respectively dissolved in 30% methanol to obtain a reference solution; the concentration of uridine in the reference solution was 40 μg / mL; the concentration of 5-hydroxymethylfurfural in the reference solution was 40 μg / mL; the concentration of daidzein in the reference solution was 40 μg / mL.

[0022] Preparation of control medicinal material solution: Polygonatum sibiricum control medicinal material is added with water and refluxed, filtered, and ultrasonicated with 30% methanol to obtain a control medicinal material solution; wherein, the mass ratio of the prepared Polygonatum sibiricum control medicinal material to water is 1:50.

[0023] The solvent of the present invention is 30% methanol. When 30% methanol is used as the extraction solvent, the amount of chromatographic peak information is large and the separation effect of each chromatographic peak is good.

[0024] The reference substance solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference substance; and the components of the HPLC characteristic spectrum of the prepared polygonatum are qualitatively measured according to the chromatogram of the reference substance.

[0025] The mobile phase A of the present invention is acetonitrile, the mobile phase B is 0.1% phosphoric acid aqueous solution, and the elution is performed in a gradient manner.

[0026] The gradient elution of the present invention is preferably specifically:

[0027] 0-3 min, phase A: 0, phase B: 100%;

[0028] 3-20 min, phase A: 0-10, phase B: 100%-90%;

[0029] 20-39 min, phase A: 10-19, phase B: 90%-81%;

[0030] 39-61 min, phase A: 19-35, phase B: 81%-65%;

[0031] 61-70min, phase A: 35, phase B: 65%.

[0032] The present invention has good baseline separation under the above elution gradient, good separation of each peak and a stable baseline.

[0033] The chromatographic column is a C18 column with specifications of 250×4.6mm, 5μm; including but not limited to ShimNex HE C18-AQ 250×4.6mm, 5μm, Shim-pack GIST C18-AQ 250×4.6mm, Ultimate AQ-C18 4.6×250mm, 5μm, or Ecosil 120-5-AQ PLUS 4.6×250mm, 5μm. Preferably, the chromatographic column is Ultimate AQ-C18 4.6×250mm, 5μm. The column temperature is 15°C to 30°C, preferably 15°C. When the column temperature is 15°C, the chromatogram peak shape is relatively symmetrical, and the resolution is good.

[0034] All the different chromatographic columns in this invention exhibited nine common peaks. The Ultimate AQ-C18 column exhibited better peak shape, so the Ultimate AQ-C18 4.6×250mm 5μm column was recommended. The theoretical plate number, calculated based on the 5-hydroxymethylfurfural peak, should be no less than 5000.

[0035] The flow rate of the mobile phase is 0.8 ml / min to 1.2 ml / min, preferably 1 ml / min.

[0036] The present invention found that the separation of each chromatographic peak was better and the peak shape was better at a flow rate of 1 ml / min, which was the most preferred solution.

[0037] The detection wavelength of the present invention is preferably 260 nm.

[0038] The inventors found that at 260 nm, the chromatographic information is rich, each component has good absorption, the response value is moderate, the chromatographic peak information volume is large, the peak separation is good, and the baseline is stable.

[0039] The injection volume of the present invention is 3 to 10 μL, preferably 5 μL.

[0040] The beneficial effects of the present invention are that, under a liquid chromatography condition, the substance group of polygonatum is controlled by fingerprint, and uridine, 5-hydroxymethylfurfural and daidzein are used to locate the fingerprint; the cost of detection can be greatly reduced, and qualitative detection can be achieved.

[0041] The similarity of the HPLC characteristic spectrum of processed Polygonatum sibiricum was evaluated using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and a HPLC standard characteristic spectrum of processed Polygonatum sibiricum consisting of 9 characteristic peaks was obtained, including peak 1 uridine, peak 4 5-hydroxymethylfurfural, and peak 8 daidzein.

[0042] In the standard characteristic spectrum of the prepared Polygonatum sibiricum formula granules, 5-hydroxymethylfurfural is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.68 (peak 1), 0.70 (peak 2), 0.80 (peak 3), 1.12 (peak 5), 1.42 (peak 6), 1.60 (peak 7), 2.49 (peak 8), and 2.82 (peak 9).

[0043] Quality judgment standard: Take a sample of processed Polygonatum sibiricum and operate according to the same method as above to obtain the characteristic spectrum of processed Polygonatum sibiricum. Use the "Similarity Evaluation System of Chinese Medicine Chromatographic Fingerprints" (2012 edition) of the National Pharmacopoeia Committee to analyze the standard characteristic spectrum of processed Polygonatum sibiricum and the sample characteristic spectrum. The similarity is greater than 0.90.

[0044] The method provided by the present invention can effectively monitor the quality of different batches of processed polygonatum, so that the quality is stable. The method has the characteristics of high precision and good reproducibility, and is conducive to comprehensive monitoring of product quality.

[0045] The characteristic spectrum of processed Polygonatum sibiricum established by the present invention uses uridine, 5-hydroxymethylfurfural and daidzein as reference substances, focuses on the order of each characteristic peak and the correlation with medicinal materials and intermediate products, can comprehensively evaluate the overall quality features of the product, and the method is scientific and reliable.

[0046] The newly developed characteristic spectrum method of the present invention can detect highly polar components in processed Polygonatum sibiricum and its standard decoction. Furthermore, the sample preparation method is simple and easy to operate, and relatively many characteristic peaks can be identified. This method enables accurate and reliable characteristic spectrum detection of processed Polygonatum sibiricum and its preparations. The authenticity, quality consistency, and stability of processed Polygonatum sibiricum and its preparations can be effectively tested and controlled. This provides a basis for effective control and comprehensive evaluation of the quality of processed Polygonatum sibiricum preparations, ensuring the uniformity and stability of the quality of processed Polygonatum sibiricum preparations.

[0047] The present invention is applicable to the detection method of the high-performance liquid phase characteristic spectrum of the processed polygonatum, can comprehensively control the characteristic components in the processed polygonatum, ensure the overall stability of the quality of the processed polygonatum, and the method is simple to operate, high in precision, good in stability, good in repeatability, and high in accuracy.

[0048] The present invention provides a method for constructing an HPLC characteristic spectrum of processed polygonatum, comprising: A) dissolving a processed polygonatum raw material in a solvent to obtain a test solution; B) measuring the test solution by high performance liquid chromatography to obtain an HPLC characteristic spectrum of the processed polygonatum raw material; the chromatographic conditions of the high performance liquid chromatography are: a C18 column; mobile phase A is acetonitrile; mobile phase B is a 0.1% phosphoric acid aqueous solution, and gradient elution is performed. The present invention uses high performance liquid chromatography, selects acetonitrile-0.1% phosphoric acid as the mobile phase for gradient elution, and uses uridine, 5-hydroxymethylfurfural, and daidzein as reference substances to establish an HPLC characteristic spectrum of processed polygonatum. The method has good repeatability and precision, is stable and reliable, and can be used to control the quality of processed polygonatum.

[0049] The invention establishes a high-performance liquid phase characteristic spectrum method for detecting prepared polygonatum slices, standard decoctions, extract formula granules and related preparations.

[0050] In the process of establishing the characteristic spectrum of processed Polygonatum odoratum, the present invention identified nine common characteristic peaks and studied their relative retention times and relative peak areas, ensuring its chemical composition stability and safety in use. A method for determining the content of processed Polygonatum odoratum was established, with three index components identified. This overcomes the drawback that single component content determination is difficult to reflect the overall content, and allows for overall and macroscopic control of the intrinsic quality of processed Polygonatum odoratum and its related preparations, ensuring the efficacy of the drugs and providing more standardized quality control for the medicinal material and its related preparations.

[0051] The method can be used to identify Polygonatum sibiricum and its processed products. The chromatogram of the processed Polygonatum sibiricum sample showed nine characteristic peaks. Except for peaks 4, 6, 8, and 9, the retention times of all the peaks corresponded to the retention times of the five characteristic peaks in the chromatogram of the Polygonatum sibiricum sample. Peak 4 (5-hydroxymethylfurfural), peak 6, peak 8 (daidzin), and peak 9 are the components of Polygonatum sibiricum after being processed into processed Polygonatum sibiricum.

[0052] The method of the present invention has good stability, high precision, good reproducibility, is convenient and easy to master. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the chromatogram of different wavelengths of the prepared Polygonatum sibiricum granules;

[0054] Figure 2 Column temperature inspection chromatogram;

[0055] Figure 3 This is the flow velocity investigation result diagram;

[0056] Figure 4 This is the delay investigation result diagram;

[0057] Figure 5 Investigation of extraction solvents;

[0058] Figure 6 Investigation of extraction methods;

[0059] Figure 7 Extraction time investigation;

[0060] Figure 8 Weighing and inspection of samples;

[0061] Figure 9 Injection volume combination chart;

[0062] Figure 10 Chromatographic peak identification;

[0063] Figure 11 Uridine spectrum - reference;

[0064] Figure 12 Uridine spectrum - processed Polygonatum formula granules;

[0065] Figure 13 5-Hydroxymethylfurfural-spectrum-reference substance;

[0066] Figure 14 Spectrum of 5-hydroxymethylfurfural - Polygonatum sibiricum formula granules;

[0067] Figure 15 Spectrum of daidzein - reference substance;

[0068] Figure 16 Spectrum of daidzein - processed Polygonatum sibiricum granules;

[0069] Figure 17 Comparison chart of different chromatographic columns;

[0070] Figure 18 Verification diagram of characteristic spectra of 3 batches of Polygonatum sibiricum formula granules;

[0071] Figure 19 Comparative characteristic spectrum of processed Polygonatum sibiricum formula granules;

[0072] Figure 20 Characteristic spectrum of processed Polygonatum sibiricum slices;

[0073] Figure 21 Characteristic spectrum of standard decoction of Polygonatum sibiricum;

[0074] Figure 22 Characteristic spectrum of processed Polygonatum sibiricum extract;

[0075] Figure 23 Characteristic spectrum of Polygonatum sibiricum medicinal material;

[0076] Figure 24 Characteristic spectrum of Polygonatum formula granules;

[0077] Figure 25 Comparison chart of the characteristics of the Huangjing formula granules and the characteristics of the processed Huangjing formula granules;

[0078] Figure 26 It is the chromatogram of Comparative Example 1;

[0079] Figure 27 It is the chromatogram of Comparative Example 2. DETAILED DESCRIPTION

[0080] The present invention provides a method for constructing an HPLC characteristic spectrum of processed Polygonatum odoratum. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted that all similar replacements and modifications are obvious to those skilled in the art and they all fall within the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0081] To further illustrate the present invention, the method for constructing an HPLC characteristic spectrum of processed Polygonatum sibiricum provided by the present invention is described in detail below in conjunction with examples.

[0082] High performance liquid chromatograph: Agilent 1260 high performance liquid chromatograph, Waters e2695 high performance liquid chromatograph, Shimadzu 20AD high performance liquid chromatograph;

[0083] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0084] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0085] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0086] Columns: Ultimate AQ-C18 250 × 4.6 mm 5 μm, ShimNex HE C18-AQ 250 × 4.6 mm 5 μm, Shim-pack GIST C18-AQ 250 × 4.6 mm, Ecosil 120-5-AQ PLUS 4.6 × 250 mm 5 μm;

[0087] Polygonatum sibiricum reference medicinal material (China Food and Drug Inspection Institute, batch number: 121341-201705);

[0088] 5-Hydroxymethylfurfural (China Food and Drug Inspection Institute, batch number 111626-202215, purity 99.5%)

[0089] Uridine (China Food and Drug Inspection Institute, batch number 110887-202104, purity 99.6%);

[0090] Daidzein (China Food and Drug Administration, batch number: 111738-201904, purity 93.4%)

[0091] Acetonitrile was of chromatographic grade; ammonium acetate was of chromatographic grade; water was ultrapure water; and other reagents were of analytical grade.

[0092] Prepared Polygonatum sibiricum formula granules (ZHJ-PFKL-01, ZHJ-PFKL-02, ZHJ-PFKL-03).

[0093] Polygonatum decoction pieces (ZHJ-YP-01, ZHJ-YP-02, ZHJ-YP-03, ZHJ-YP-04, ZHJ-YP-05, ZHJ-YP-06, ZHJ-YP-07, ZH J-YP-08, ZHJ-YP-09, ZHJ-YP-10, ZHJ-YP-11, ZHJ-YP-12, ZHJ-YP-13, ZHJ-YP-14, ZHJ-YP-15)

[0094] Standard decoctions of Polygonatum sibiricum (ZHJ-BT-01, ZHJ-BT-02, ZHJ-BT-03, ZHJ-BT-04, ZHJ-BT-05, ZHJ-BT-06, ZHJ-BT-07, ZHJ-BT-08, ZHJ-BT-09, ZHJ-BT-10, ZHJ-BT-11, ZHJ-BT-12, ZHJ-BT-13, ZHJ-BT-14, ZHJ-BT-15)

[0095] Prepared Polygonatum sibiricum Extract (ZHJ-TWQ-01, ZHJ-TWQ-02, ZHJ-TWQ-03)

[0096] Polygonatum sibiricum medicinal materials (HJ01, HJ02, HJ03, HJ04, HJ05, HJ06, HJ07, HJ08, HJ09, HJ10, HJ11, HJ12, HJ13, HJ14, HJ15)

[0097] Polygonatum sibiricum formula granules (HJ-PFKL-01, HJ HJ-PFKL-02, HJ-PFKL-03).

[0098] Example 1 Characteristic Spectrum Method for Processing Polygonatum Formula Granules

[0099] 1.1 Chromatographic Conditions and System Suitability Test: Octadecylsilane bonded silica gel was used as the filler (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); acetonitrile was used as mobile phase A; 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed as specified in the table below; the detection wavelength was 260 nm; the flow rate was 1.0 ml / min, and the column temperature was 15°C. The number of theoretical plates, calculated based on the 5-hydroxymethylfurfural peak, should be no less than 5000.

[0100]

[0101] Preparation of Reference Solution: Take 1g of Polygonatum sibiricum reference medicinal material, add 50ml of water, decoct for 30 minutes, centrifuge, take the supernatant and evaporate to dryness. Add 5ml of 30% methanol to the residue and ultrasonicate (power 600W, frequency 40kHz) for 20 minutes. Let cool, shake well, filter, and take the filtrate as the reference medicinal material solution. Take appropriate amounts of uridine reference substance, 5-hydroxymethylfurfural reference substance, and daidzein reference substance, add 30% methanol to make a mixed solution containing 40μg per 1ml, which is used as the reference substance solution.

[0102] Preparation of the test solution: Take an appropriate amount of prepared Polygonatum sibiricum granules, grind them into powder, take about 0.5 g, accurately weigh, place in a stoppered conical flask, accurately add 10 ml of 30% methanol, treat with ultrasound (power 600 W, frequency 40 kHz) for 20 minutes, let cool, shake well, filter, and take the filtrate.

[0103] Determination method: Accurately aspirate 5μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0104] 1.2 Chromatographic conditions and system suitability test

[0105] 1.2.1 Wavelength Selection

[0106] Based on the experimental conditions proposed above, the diode array detector was used to scan the entire wavelength range of the test solution, and the chromatograms of the test solution at wavelengths of 220 nm, 240 nm, 260 nm, and 280 nm were extracted respectively. Figure 1 . Figure 1 These are chromatograms of different wavelengths of the prepared Polygonatum sibiricum granules.

[0107] The results showed that the chromatographic peak information content was larger and the chromatogram baseline was more stable when the detection wavelength was 260 nm, so the detection wavelength was determined to be 260 nm.

[0108] 1.2.2 Column temperature investigation

[0109] Based on the experimental conditions proposed above, the column temperatures of 15℃, 20℃, 25℃ and 30℃ were investigated. Figure 2 . Figure 2 Column temperature inspection chromatogram.

[0110] The results of column temperature investigation showed that when the column temperature was 15℃, the chromatogram peaks were relatively symmetrical and the separation was good, so 15℃ was finally determined as the column temperature for the characteristic spectrum method of Polygonatum sibiricum granules.

[0111] 1.2.3 Flow rate investigation

[0112] Based on the experimental conditions proposed above, the flow rates of 0.8ml / min, 1.0ml / min, and 1.2ml / min were investigated. Figure 3 . Figure 3 This is the flow rate investigation result diagram.

[0113] The results showed that when the flow rate was 1.0 ml / min, the chromatogram peak shape was better and the separation was moderate. Therefore, the flow rate was determined to be 1.0 ml / min.

[0114] 1.2.4 Delay Investigation

[0115] Based on the experimental conditions proposed above, a delay test was conducted. The results are shown in Figure 4 . Figure 4 This is a graph showing the delay investigation results.

[0116] The results showed that the sample had basically no chromatographic peak after 70 minutes, so the sample detection time was set at 70 minutes.

[0117] In summary, the chromatographic conditions and system usability test for the characteristic spectrum of the prepared Polygonatum sibiricum granules are tentatively determined as follows: octadecylsilane bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A; 0.1% phosphoric acid as mobile phase B, gradient elution as specified in the table below; detection wavelength 260 nm; flow rate 1.0 ml / min; column temperature 15°C. The number of theoretical plates calculated based on the 5-hydroxymethylfurfural peak should be no less than 5000.

[0118]

[0119] 1.2.5 Preparation of test solution

[0120] 1.2.5.1 Extraction solvent investigation

[0121] Take an appropriate amount of this product, grind it into powder, take about 0.5g, weigh it accurately, put it into a stoppered conical flask, and accurately add 30% ethanol, 70% ethanol, ethanol, 30% methanol, 70% methanol, methanol, and 10ml of water respectively. Treat it with ultrasound (power 600W, frequency 40kHz) for 20 minutes, let it cool, shake it well, filter it, and take the filtrate. The results are shown in the figure. Figure 5 . Figure 5Investigation of extraction solvents.

[0122] From the above, it can be seen that when 30% methanol is used as the extraction solvent, the chromatographic peak information volume is large and the separation effect of each chromatographic peak is good, so the extraction solvent is determined to be 30% methanol.

[0123] 1.2.5.2 Investigation of extraction methods

[0124] Take an appropriate amount of this product, grind it into powder, take about 0.5g, weigh it accurately, put it into a stoppered conical flask, add 10ml of 30% methanol, ultrasonically treat it (power 600W, frequency 40kHz) for 20 minutes, reflux it for 20 minutes, let it cool, shake it well, filter it, and take the filtrate. Figure 6 . Figure 6 Investigation of extraction methods.

[0125] The results showed that the chromatographic peak information of ultrasonic and reflux extraction was basically consistent, so the extraction method of the test sample was determined to be ultrasonic extraction, which was simpler to operate.

[0126] 1.2.5.3 Extraction time investigation

[0127] Take an appropriate amount of this product, grind it into powder, take about 0.5g, weigh it accurately, put it into a stoppered conical flask, add 10ml of 30% methanol, and ultrasonically treat it (power 600W, frequency 40kHz) for 20, 30, and 40 minutes respectively. Let it cool, shake it well, filter it, and take the filtrate. Figure 7 . Figure 7 Extraction time investigation.

[0128] As can be seen from the figure, sufficient extraction can be achieved when the extraction time is 20 minutes, so the extraction time of the test sample is determined to be 20 minutes.

[0129] 1.2.5.4 Sample weighing inspection

[0130] Take an appropriate amount of this product, grind it into powder, take about 0.3g, 0.5g, and 1.0g, weigh accurately, place in a stoppered conical flask, accurately add 10ml of 30% methanol, treat with ultrasound (power 600W, frequency 40kHz) for 20 minutes, let cool, shake well, filter, and take the filtrate. Figure 8 . Figure 8 Weighing and inspection of samples.

[0131] From the above, we can see that when the sample weight is 0.5g, the characteristic spectrum chromatographic peak area is moderate and the baseline is more stable. Therefore, the sample weight is determined to be 0.5g.

[0132] 1.2.5.5 Determine the test sample preparation method

[0133] Take an appropriate amount of this product, grind it into powder, take about 0.5g, weigh it accurately, put it into a stoppered conical flask, add 10ml of 30% methanol, ultrasonically treat it (power 600W, frequency 40kHz) for 20 minutes, let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0134] 1.2.6 Determination method

[0135] 1.2.6.1 Sample injection volume investigation

[0136] Prepare the test solution according to the above determined test solution preparation method, accurately draw 3μl, 5μl, and 10μl of the test solution respectively, inject it into the liquid chromatograph, and measure it. Figure 9 . Figure 9 Injection volume combination chart.

[0137] As can be seen from the figure, when the injection volume is 5μl, the peak areas in the chromatogram are moderate, so the injection volume is determined to be 5μl.

[0138] The determination method is as follows: accurately aspirate 5 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine them.

[0139] Example 2 Characteristic Spectrum Method Tentative

[0140] 2.1 Chromatographic Conditions and System Suitability Test: Octadecylsilane bonded silica gel (Ultimate AQ-C18, 4.6 mm × 250 mm, 5 μm, or a column of equivalent performance) was used as the packing material. Acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B. Gradient elution was performed as specified in the table below. The detection wavelength was 260 nm. The flow rate was 1.0 ml / min. The column temperature was 15°C. The number of theoretical plates, calculated based on the 5-hydroxymethylfurfural peak, should be no less than 5000.

[0141]

[0142] Preparation of Reference Solution: Take 1g of Polygonatum sibiricum reference medicinal material and add 50ml of water. Heat and reflux extraction for 30 minutes, centrifuge, and evaporate the supernatant to dryness. Add 5ml of 30% methanol to the residue and ultrasonically treat (power 600W, frequency 40kHz) for 20 minutes. Cool, shake well, filter, and collect the filtrate as the reference medicinal material solution. Take appropriate amounts of uridine reference substance, 5-hydroxymethylfurfural reference substance, and daidzein reference substance and add 30% methanol to prepare a mixed solution containing 40μg per 1ml.

[0143] Preparation of the test solution: Take an appropriate amount of prepared Polygonatum sibiricum granules, grind them into powder, take about 0.5 g, accurately weigh, place in a stoppered conical flask, accurately add 10 ml of 30% methanol, treat with ultrasound (power 600 W, frequency 40 kHz) for 20 minutes, let cool, shake well, filter, and take the filtrate.

[0144] Determination method: Accurately aspirate 5μl of reference solution and test solution, inject into liquid chromatograph, and determine.

[0145] 2.2 Methodological Investigation

[0146] 2.2.1 Chromatographic peak identification

[0147] Preparation of test solution: Take an appropriate amount of prepared Polygonatum sibiricum granules, grind them into powder, take about 0.5g, accurately weigh, place in a stoppered conical flask, accurately add 10ml of 30% methanol, treat with ultrasound (power 600W, frequency 40kHz) for 20 minutes, cool, shake well, filter, and take the filtrate.

[0148] Preparation of reference medicinal material solution: Take 1 g of Polygonatum sibiricum reference medicinal material, add 50 ml of water, decoct for 30 minutes, centrifuge, take the supernatant and evaporate to dryness, add 5 ml of 30% methanol to the residue, treat with ultrasound (power 600 W, frequency 40 kHz) for 20 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material solution.

[0149] Preparation of reference substance solution: Take appropriate amount of uridine reference substance, 5-hydroxymethylfurfural reference substance, and daidzein reference substance, add 30% methanol to make a mixed solution containing 40 μg per 1 ml, which is used as the reference substance solution.

[0150] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution lacking processed Polygonatum sibiricum granules was prepared.

[0151] Position the characteristic spectrum peaks of the prepared Polygonatum sibiricum granules, see Figure 10-16 . Figure 10 Chromatographic peak identification; Figure 11 Uridine spectrum - reference; Figure 12 Uridine spectrum - processed Polygonatum sibiricum formula granules; Figure 13 5-Hydroxymethylfurfural-spectrum-reference substance; Figure 14 Spectrum of 5-hydroxymethylfurfural - Polygonatum sibiricum formula granules; Figure 15 Spectrum of daidzein - reference substance; Figure 16 Spectrum of daidzein - processed Polygonatum sibiricum formula granules.

[0152] The results showed that among the 9 characteristic peaks of the test sample, peak 1 was uridine, peak 4 was 5-hydroxymethylfurfural, and peak 8 was daidzein. In the following methodological investigation, the 9 characteristic peaks in the test sample were investigated.

[0153] 2.2.2 Precision test

[0154] Prepare one aliquot of the test solution according to the proposed experimental method, inject the sample six times continuously, and calculate the retention time of each characteristic peak. See Table 1.

[0155] Table 1 Precision investigation-retention time

[0156]

[0157] It can be seen from the table that the instrument precision of this method is good.

[0158] 2.2.3 Repeatability study

[0159] Prepare 6 test sample solutions according to the proposed experimental method, measure, and calculate the relative retention time and relative peak area of ​​each characteristic peak. See Table 2.

[0160] Table 2 Repeatability study - relative retention time ratio

[0161]

[0162]

[0163] From the above, we can see that this method has good repeatability.

[0164] 2.2.4 Intermediate precision study

[0165] At time T1, person A1 prepared one aliquot of the test solution according to the proposed experimental method and measured it on instrument C1 (Agilent 1260). Person A2 prepared one aliquot of the test solution according to the proposed experimental method at time T2 and measured it on instrument C2 (Shimadzu LC-20AD). The relative retention time and relative peak area of ​​each characteristic peak were calculated. See Table 3.

[0166] Table 3 Intermediate precision investigation - relative retention time ratio

[0167]

[0168] From the above, we can see that this method has good durability on Agilent instruments.

[0169] 2.2.5 Durability inspection

[0170] 2.2.5.1 Column durability assessment

[0171] Based on the experimental conditions proposed above, the chromatographic columns ShimNex HE C18-AQ 250×4.6mm 5μm, Shim-pack GIST C18-AQ 250×4.6mm, Ultimate AQ-C184.6×250mm 5μm, and Ecosil 120-5-AQ PLUS 4.6×250mm5μm were investigated. See Table 4. Figure 17 . Figure 17 Comparison chart of different chromatographic columns.

[0172] Table 4 Column durability investigation - relative retention time

[0173]

[0174]

[0175] As can be seen from the figure above, nine common peaks appear on the ShimNex HE C18-AQ 250×4.6mm5μm, Shim-pack GIST C18-AQ 250×4.6mm, Ultimate AQ-C18 4.6×250mm 5μm, and Ecosil 120-5-AQ PLUS 4.6×250mm 5μm columns. The Ultimate AQ-C18 4.6×250mm 5μm column has the best separation effect and is recommended.

[0176] 2.2.5.2 Stability

[0177] According to the experimental conditions proposed above, prepare a sample solution and measure it at 0h, 4h, 8h, 12h, 16h, 20h, and 24h. See Table 5.

[0178] Table 5 Stability Study-Retention Time

[0179]

[0180] As can be seen from the table, the RSD of the characteristic peak retention time is 0.07% to 0.20%, and the sample solution is relatively stable within 24 hours.

[0181] 2.2.6 Determination of characteristic peaks and establishment of reference spectrum

[0182] 2.2.6.1 Verification results of three batches of Polygonatum sibiricum granules

[0183] The proposed method was used to determine the characteristic spectra of three batches of samples of this product and calculate the relative retention time. Figure 18 , Table 6. Figure 18 Verification diagram of characteristic spectra of 3 batches of Polygonatum sibiricum formula granules.

[0184] Table 6 Relative retention time of three batches of Polygonatum sibiricum granules

[0185]

[0186]

[0187] Nine robust peaks were selected as characteristic peaks based on relative retention times and consistent detection across all sample batches at relatively high levels. Based on the methodological review and validation results from three batches of particles, the number of theoretical plates, calculated based on 5-hydroxymethylfurfural, was tentatively determined to be no less than 5,000.

[0188] 2.2.6.2 Establishment of relative retention time limit values

[0189] The summary of the methodological investigation items and verification results is shown in Table 7.

[0190] Table 7 Summary of RSD% results for each item of the methodology - retention time / relative retention time

[0191]

[0192] As can be seen from the table, the RSD values ​​of the relative retention times of the characteristic peaks are small and within the range of ±10% of the average value, so the specified value range of the relative retention time of each peak is temporarily set at ±10%.

[0193] In summary, the regulations should be as follows: the test sample chromatogram should show 9 characteristic peaks. Except for peaks 4, 6, 8, and 9, the retention times of all the peaks should correspond to the retention times of the 5 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the peak of the 5-hydroxymethylfurfural reference material is the S peak. The relative retention time of each characteristic peak and the S peak should be calculated. The relative retention time should be within ±10% of the specified value, which is: 0.68 (peak 1), 0.70 (peak 2), 0.80 (peak 3), 1.12 (peak 5), 1.42 (peak 6), 1.60 (peak 7), 2.49 (peak 8), and 2.82 (peak 9).

[0194] Three batches of Polygonatum sibiricum granules were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison spectrum of the characteristic spectrum of Polygonatum sibiricum granules was established. Figure 19 . Figure 19 Comparative characteristic spectrum of prepared Polygonatum sibiricum granules; Peak 1: uridine; Peak 4 (S): 5-hydroxymethylfurfural; Peak 8: daidzein; Chromatographic column: Ultimate AQ-C18, 4.6 mm × 250 mm, 5 μm.

[0195] Example 3 Decoction pieces

[0196] The reference solution of the reference medicinal material was prepared in the same way as the reference solution of the characteristic spectrum method. The test solution of the prepared Polygonatum sibiricum slices was prepared and 15 batches of prepared Polygonatum sibiricum slices were tested. The characteristic spectrum of the 15 batches of prepared Polygonatum sibiricum slices is shown in Figure 24 The relative retention time results and chromatogram results of each characteristic peak are shown in Table 8. Figure 20 . Figure 20Characteristic spectrum of prepared Polygonatum sibiricum slices; Peak 1: uridine; Peak 4 (S): 5-hydroxymethylfurfural; Peak 8: daidzein; (S1-S15 are: ZHJ-YP-01, ZHJ-YP-02, ZHJ-YP-03, ZHJ-YP-04, ZHJ-YP-05, ZHJ-YP-06, ZHJ-YP-07, ZHJ-YP-08, ZHJ-YP-09, ZHJ-YP-10, ZHJ-YP-11, ZHJ-YP-12, ZHJ-YP-13, ZHJ-YP-14, ZHJ-YP-15).

[0197] Table 8 Relative retention time of characteristic spectra of 15 batches of prepared Polygonatum sibiricum slices

[0198]

[0199] The chromatogram of the test sample showed nine characteristic peaks. Except for peaks 4, 6, 8, and 9, the retention times of all of them corresponded to the retention times of the five characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the peak of the 5-hydroxymethylfurfural reference material was the S peak. The relative retention times of the characteristic peaks and the S peak were calculated, and the relative retention times were all within the range of ±10% of the specified values, which were 0.67 (peak 1), 0.73 (peak 2), 0.78 (peak 3), 1.12 (peak 5), 1.44 (peak 6), 1.62 (peak 7), 2.54 (peak 8), and 2.88 (peak 9). Therefore, this method can be applied to the detection of characteristic chromatograms of prepared Polygonatum sibiricum slices.

[0200] Example 4 Standard Decoction

[0201] The test solution of standard decoction of Polygonatum odoratum was prepared in the same way as the preparation method of the test solution according to the characteristic spectrum method, and 15 batches of standard decoction of Polygonatum odoratum were tested. The characteristic spectrum of 15 batches of standard decoction of Polygonatum odoratum is shown in Figure 24 The relative retention time results and chromatogram results of each characteristic peak are shown in Table 9. Figure 21 . Figure 21 Characteristic spectrum of the standard decoction of Polygonatum sibiricum; Peak 1: uridine; Peak 4 (S): 5-hydroxymethylfurfural; Peak 8: daidzein (batch numbers from bottom to top: ZHJ-BT-01, ZHJ-BT-02, ZHJ-BT-03, ZHJ-BT-04, ZHJ-BT-05, ZHJ-BT-06, ZHJ-BT-07, ZHJ-BT-08, ZHJ-BT-09, ZHJ-BT-10, ZHJ-BT-11, ZHJ-BT-12, ZHJ-BT-13, ZHJ-BT-14, ZHJ-BT-15).

[0202] Table 9 Relative retention time of characteristic spectra of 15 batches of standard decoction of Polygonatum odoratum

[0203]

[0204] The chromatogram of the test sample showed nine characteristic peaks. Except for peaks 4, 6, 8, and 9, the retention times of all of them corresponded to the retention times of five characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the peak of the 5-hydroxymethylfurfural reference material was the S peak. The relative retention times of each characteristic peak and the S peak were calculated and all within ±10% of the specified values: 0.68 (peak 1), 0.73 (peak 2), 0.79 (peak 3), 1.00 (peak 4), 1.12 (peak 5), 1.48 (peak 6), 1.62 (peak 7), 2.53 (peak 8), and 2.87 (peak 9). Therefore, this method can be applied to the detection of the characteristic chromatogram of the standard decoction of Polygonatum sibiricum.

[0205] Example 5 Extract

[0206] The test solution of prepared polygonatum extract was prepared in the same way as the test solution of the characteristic spectrum method, and 3 batches of prepared polygonatum extract were tested. The characteristic spectra of the 3 batches of prepared polygonatum extract are shown in Figure 24 The relative retention time results and chromatogram results of each characteristic peak are shown in Table 10. Figure 22 . Figure 22 Characteristic spectrum of processed Polygonatum sibiricum extract; Peak 1: uridine; Peak 4 (S): 5-hydroxymethylfurfural; Peak 8: daidzein; (Batch numbers from bottom to top: ZHJ-TWQ-01, ZHJ-TWQ-02, ZHJ-TWQ-03).

[0207] Table 10 Relative retention time of characteristic spectra of three batches of Polygonatum sibiricum extracts

[0208]

[0209]

[0210] The test sample chromatogram showed nine characteristic peaks. Except for peaks 4, 6, 8, and 9, the retention times of all of them corresponded to the retention times of five characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the peak of the 5-hydroxymethylfurfural reference material was the S peak. The relative retention times of each characteristic peak and the S peak were calculated and all within ±10% of the specified values: 0.68 (peak 1), 0.70 (peak 2), 0.79 (peak 3), 1.12 (peak 5), 1.42 (peak 6), 1.60 (peak 7), 2.48 (peak 8), and 2.81 (peak 9). Therefore, this method can be applied to the detection of the characteristic spectrum of the Polygonatum sibiricum extract.

[0211] Comparison of Example 6 with Polygonatum odoratum medicinal materials and Polygonatum odoratum formula granules

[0212] 6.1 Polygonatum sibiricum

[0213] According to the method for preparing the control medicinal material reference solution of the characteristic spectrum method of Example 1 of the present invention, the polygonatum medicinal material test solution was prepared in the same manner, and 15 batches of polygonatum medicinal materials were tested. The characteristic spectrum of 15 batches of polygonatum medicinal materials is shown in FIG. Figure 24 The relative retention time results and chromatogram results of each characteristic peak are shown in Table 11. Figure 23 . Figure 23 Characteristic spectrum of Polygonatum sibiricum medicinal material; Peak 1 (S): Uridine (Batch numbers from bottom to top are: HJ01, HJ02, HJ03, HJ04, HJ05, HJ06, HJ07, HJ08, HJ09, HJ10, HJ11, HJ12, HJ13, HJ14, HJ15)

[0214] Table 11 Relative retention time of characteristic spectra of 15 batches of Polygonatum sibiricum

[0215]

[0216] 6.2 Polygonatum formula granules

[0217] The test solution of Polygonatum sibiricum formula granules was prepared in the same way as the test solution preparation method of the characteristic spectrum method, and 3 batches of Polygonatum sibiricum formula granules were tested. The characteristic spectra of the 3 batches of Polygonatum sibiricum formula granules are shown in Figure 24 The relative retention time results and chromatogram results of each characteristic peak are shown in Table 12. Figure 24 . Figure 24 Characteristic spectrum of Polygonatum sibiricum formula granules; Peak 1 (S): Uridine; (Batch numbers from bottom to top: HJ-PFKL-01, HJ HJ-PFKL-02, HJ-PFKL-03)

[0218] Table 12 Relative retention times of characteristic spectra of three batches of Polygonatum sibiricum granules

[0219]

[0220] 6.3 Comparison of Characteristic Spectra of Polygonatum Formula Granules and Processed Polygonatum Formula Granules

[0221] Three batches of Polygonatum sibiricum formula granules were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a reference spectrum of the characteristic spectrum of Polygonatum sibiricum formula granules was established, which was compared with the reference spectrum of the characteristic spectrum of the processed Polygonatum sibiricum formula granules. Figure 25 . Figure 25 Comparison of the characteristic reference spectrum of Polygonatum sibiricum formula granules and the characteristic reference spectrum of processed Polygonatum sibiricum formula granules; Peak 1: uridine; Peak 4 (S): 5-hydroxymethylfurfural; Peak 8: daidzein.

[0222] The results showed that this method can be used to identify Polygonatum sibiricum and its processed products. The chromatogram of the processed Polygonatum sibiricum sample showed nine characteristic peaks. Except for peaks 4, 6, 8, and 9, the retention times of all the peaks corresponded to the five characteristic peaks in the chromatogram of the Polygonatum sibiricum preparation. Peak 4 (5-hydroxymethylfurfural), peak 6, peak 8 (daidzin), and peak 9 are the components of Polygonatum sibiricum after it is processed into processed Polygonatum sibiricum.

[0223] Comparative Example 1

[0224] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); acetonitrile as mobile phase A; 0.1% phosphoric acid solution as mobile phase B; gradient elution was performed as specified in the table below; and the detection wavelength was 260 nm.

[0225]

[0226] The results are as follows Figure 26 As stated, Figure 26 The chromatogram of comparative example 1 shows that the method partially has peak-envelope phenomenon.

[0227] Comparative Example 2

[0228] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.1% formic acid solution (25:75) as the mobile phase, and the detection wavelength was 260 nm. Figure 27 As shown, Figure 27 This is the chromatogram of Comparative Example 2; the results show that this method has poor chromatographic peak separation and peak wrapping phenomenon.

[0229] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing an HPLC characteristic spectrum of prepared Polygonatum odoratum, comprising: A) extracting the prepared polygonatum raw material with a solvent to obtain a test solution; the solvent is 30% methanol; the extraction method is ultrasonic extraction or heating reflux extraction; the prepared polygonatum is prepared in accordance with the Sichuan Province Traditional Chinese Medicine Preparation Specifications 2015 Edition; B) using high performance liquid chromatography to measure the test solution to obtain an HPLC characteristic spectrum of the raw material of Polygonatum sibiricum; further comprising preparing a reference substance solution and a reference medicinal material solution; Preparation of reference substance solution: uridine, 5-hydroxymethylfurfural, and daidzein were respectively dissolved in 30% methanol to obtain a reference substance solution; the concentration of daidzein in the reference substance solution was 40 μg / mL; the concentration of uridine in the reference substance solution was 40 μg / mL; the concentration of 5-hydroxymethylfurfural in the reference substance solution was 40 μg / mL; Preparation of control medicinal material solution: using Polygonatum sibiricum control medicinal material, adding water to reflux, filtering, evaporating the filtrate to dryness, adding 30% methanol to sonicate the residue to obtain a control medicinal material solution; The reference substance solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference substance; and the components of the HPLC characteristic spectrum of the prepared Polygonatum sibiricum are qualitatively determined according to the chromatogram of the reference substance; The HPLC conditions are as follows: a C18 column; mobile phase A is acetonitrile; mobile phase B is a 0.1% phosphoric acid aqueous solution; gradient elution; detection wavelength is 260 nm; The gradient elution is specifically as follows: 0-3 min, phase A: 0%, phase B: 100%; 3-20 min, phase A: 0%-10%, phase B: 100%-90%; 20-39 min, phase A: 10%-19%, phase B: 90%-81%; 39-61 min, phase A: 19%-35%, phase B: 81%-65%; 61~70min, phase A: 35%, phase B: 65%.

2. The method according to claim 1, characterized in that The chromatographic column is C18 with a specification of 250×4.6 mm 5 μm; the column temperature is 15° C., and the theoretical plate number calculated based on the 5-hydroxymethylfurfural peak should be no less than 5000.

3. The method according to claim 2, characterized in that The flow rate of the mobile phase was 1.0 mL / min; the injection volume was 5 μL.

4. The method according to claim 1, wherein The similarity of the HPLC characteristic spectrum of processed Polygonatum sibiricum was evaluated using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and a HPLC standard characteristic spectrum of processed Polygonatum sibiricum consisting of 9 characteristic peaks was obtained, including peak 1 uridine, peak 4 5-hydroxymethylfurfural, and peak 8 daidzein.

5. The method according to claim 4, characterized in that In the standard characteristic spectrum of the prepared Polygonatum sibiricum formula granules, 5-hydroxymethylfurfural is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.68 (peak 1), 0.70 (peak 2), 0.80 (peak 3), 1.12 (peak 5), 1.42 (peak 6), 1.60 (peak 7), 2.49 (peak 8), and 2.82 (peak 9).

6. The method according to claim 1, characterized in that Step A) The ultrasonic power is 600W and the frequency is 40kHz; the extraction time is 20-30min.

7. The method according to claim 1, characterized in that Step A) The ratio of the mass g of the prepared Polygonatum odoratum raw material to the volume mL of the solvent is (0.5-1): (10-50); The raw materials for preparing polygonatum include prepared polygonatum granules, decoction pieces, extracts or decoctions.

8. A method for identifying Polygonatum sibiricum and processed Polygonatum sibiricum, characterized in that: The identification is performed using the method described in any one of claims 1 to 7.

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