CN116223703A

CN116223703A - Method for establishing multi-element fingerprint of rhizoma polygonati-based original species

Info

Publication number: CN116223703A
Application number: CN202310211337.6A
Authority: CN
Inventors: 张亚中; 蒲婧哲; 胡冲; 刁卓; 徐凡
Original assignee: Anhui Food And Drug Inspection And Research Institute Anhui National Agricultural And Sideline Processed Food Quality Supervision And Inspection Center
Current assignee: Anhui Food And Drug Inspection And Research Institute Anhui National Agricultural And Sideline Processed Food Quality Supervision And Inspection Center
Priority date: 2023-03-07
Filing date: 2023-03-07
Publication date: 2023-06-06

Abstract

The invention provides a method for establishing a multi-element fingerprint of a Polygonatum sibiricum original species. The fingerprint spectrum establishment method comprises the following steps: (1) preparation of a reference substance solution; (2) preparation of a sample solution; and (3) establishing a fingerprint. The method combines the HILIC-HPLC-ELSD zymolyte fingerprint spectrum to establish a multiple fingerprint spectrum for evaluating the quality of rhizoma polygonati polysaccharide (PCP). The similarity evaluation of 34 batches of Polygonatum sibiricum polysaccharide from different sources shows that. The chromatographic data of various PCPs were fused, processed and analyzed using chemometrics methods, including HCA, PCA and PLS-DA. The method has the advantages of good separation degree, good peak shape and high resolution.

Description

Method for establishing multi-element fingerprint of rhizoma polygonati-based original species

Technical Field

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for establishing a multi-element fingerprint of a rhizoma polygonati-based original species.

Background

The sources specified by the sealwort received in the 2015 edition of the Chinese pharmacopoeia are as follows: the product is dried rhizome of Polygonatum kingianum Coll et Hemsl. According to different shapes, it is known as "big rhizoma Polygonati", "chicken head rhizoma Polygonati" and "Jiang Xing rhizoma Polygonati". The Chinese medicinal materials which are the same as the food and the Chinese medicinal materials according to the tradition are identified by the national health and family planning committee, have the effects of tonifying qi and nourishing yin, strengthening spleen, moistening lung, tonifying kidney and the like, are commonly used for resisting fatigue, regulating immunity, reducing blood sugar, reducing blood fat and the like, and have extremely high medicinal health care value. Since Polygonatum sibiricum has definite efficacy, good market acceptance and higher price, other plants of Polygonatum, such as Hubei Polygonatum sibiricum, sichuan Polygonatum sibiricum, etc., commonly used in the market are sold as Polygonatum sibiricum, and the development of the Polygonatum sibiricum industry is seriously affected. An important reason for the impossibility of the impersonation phenomenon is that the standard of the rhizoma polygonati is not clear enough, the method specificity is poor, and the quality of rhizoma polygonati of different sources is difficult to evaluate rapidly through the existing standard of the medicinal material, so that research is urgently needed around the technical standard of the rhizoma polygonati Chinese medicinal material to form a standard with more definiteness and more differentiation.

In addition, according to researches, the types of sugar in the rhizoma polygonati are rich, but the content distribution of the sugar is not uniform, and polysaccharide is an important means for evaluating the quality control of rhizoma polygonati polysaccharide by taking the polysaccharide as a main chemical component of the rhizoma polygonati, and measuring the content and judging the purity of the polysaccharide. However, the phenol-sulfuric acid method is often adopted, the accuracy is low, and the structural information of the polysaccharide cannot be obtained, so that the quality of the polygonatum polysaccharide from different sources in the market cannot be effectively evaluated. Although the partial acid hydrolysis method is simple and easy at present, the partial acid hydrolysis method is widely applied to the degradation of the polysaccharide in the traditional Chinese medicine. However, compared with enzymolysis, partial acid hydrolysis products are generally complex and have poor uniformity. The enzymatic hydrolysis condition is mild and the specificity is strong.

For comparison, the name is PMP-HPLC and chemometric based Polygonatum original species polysaccharide differential analysis, the method adopts a water extraction and alcohol precipitation method to extract polysaccharide in Polygonatum sibiricum medicinal material, and after trifluoroacetic acid (TFA) hydrolysis and 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization, HPLC is adopted to establish chromatographic fingerprint of 3 Polygonatum sibiricum; and the fingerprint is analyzed by using Similarity (SA) analysis, clustering analysis (HCA) and Principal Component Analysis (PCA), and the difference of polysaccharide of 3 primitive Polygonatum species is researched, but the method has the following defects: (1) The pre-column derivatization reaction is carried out by adopting a 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization agent, so that excessive substance components can be caused, the adverse effects on the column and the detection exist, and the precision and the controllability are poor; (2) The method has the advantages of complex sample preparation process, long time and high cost; (3) From the research of polysaccharide chromatographic fingerprint cluster analysis, the polysaccharide component differences of Polygonatum kingianum, polygonatum cyrtonema and Polygonatum cyrtonema are obvious, and the polysaccharide fingerprint of Polygonatum cyrtonema and Polygonatum cyrtonema is slightly smaller than that of Polygonatum cyrtonema, and the polysaccharide fingerprint differences of Polygonatum cyrtonema and Polygonatum cyrtonema cannot be distinguished through cluster analysis; inconsistent with the principal component analysis conclusion; the method has poor reliability.

Therefore, the research on the traditional Chinese medicine polysaccharide based on the enzymolysis mode has more directionality, purpose and controllability. The present study further investigated the enzymatic hydrolysis products of Polygonatum sibiricum polysaccharide using HPLC-HILIC-ELSD. In addition, the HILIC mode combines the ELSD technology to separate and detect the oligosaccharide, and as a result, all peaks are separated, the peak shape is good, and the resolution is high. And the accuracy and the reliability of the method are verified by adopting chemometric methods, including HCA (systematic cluster analysis), PCA (principal component analysis) and PLS-DA (partial least squares discriminant analysis), to fuse, process and analyze the chromatographic data of a plurality of Polygonatum sibiricum polysaccharides (PCPs).

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for establishing a multi-element fingerprint of a rhizoma polygonati-based original species and identifying a chemical mode, which is simple, convenient, accurate and reliable to operate; the polygonatum polysaccharide is hydrolyzed by adopting an enzymolysis method, fructosidase suitable for the polygonatum is screened out, and enzymolysis conditions and methods are optimized; the specific oligosaccharide fragments in the polygonatum polysaccharide are detected, and the hydrolysis reaction has mild enzymatic hydrolysis conditions, strong specificity and better directivity, purpose and controllability; an evaporation light detector which is more suitable for the separation of oligosaccharide fragments in polysaccharide is adopted, and is combined with an HILIC chromatographic column, and the fingerprint of the final rhizoma polygonati polysaccharide can reach 17 chromatographic peaks; the fingerprint of the polygonatum polysaccharide is constructed, and the fingerprint is stable and reliable through the verification of methodology and chemometry; solves the identification problem of the easy-to-mix rhizoma polygonati products such as Sichuan rhizoma polygonati, hubei rhizoma polygonati and the like in the current market to a certain extent.

The invention is realized by the following technical scheme:

a method for establishing a multi-element fingerprint of a Polygonatum sibiricum original species comprises the following steps:

(1) Preparation of a control solution: taking a proper amount of fructose, sucrose and glucose reference substances, precisely weighing, transferring to a volumetric flask, adding water, and fixing the volume to a scale;

(2) Preparation of test solution: mixing appropriate amount of rhizoma Polygonati polysaccharide with fructosidase solution, hydrolyzing in an oscillator, heating, centrifuging, collecting supernatant, drying, and dissolving in acetonitrile/water solution;

(3) Establishing a fingerprint spectrum: detecting the reference substance solution and the test substance solution by adopting a high performance liquid chromatograph, recording chromatograms, and establishing fingerprint patterns of the reference substance solution and the test substance solution; the chromatographic conditions are as follows:

chromatographic column: hypersil GOLD ^TM PEI HILIC HPLC, model is: 5 μm,250 mm. Times.4.6 mm; mobile phase: acetonitrile is taken as a mobile phase A, water is taken as a mobile phase B, and gradient elution is carried out; column temperature: 35 ℃; flow rate: 1.0 mL/min ^-1 The method comprises the steps of carrying out a first treatment on the surface of the A detector: detecting evaporation light; the sample injection amount is 10 mu L;

gradient elution conditions: 0-25 min, 85-65% of A; 25-35 min, 65-50% of A.

The preparation of the reference substance solution in the step (1) comprises the following steps: taking 10mg of fructose, sucrose and glucose reference substances respectively, precisely weighing, transferring into a10 mL volumetric flask, and fixing the volume of water to a scale.

The preparation of the sample solution in the step (2) comprises the following steps: adding water into rhizoma Polygonati polysaccharide to obtain rhizoma Polygonati polysaccharide solution with concentration of 0.5mg/mL, adding fructosidase solution with equal volume, mixing, placing into an oscillator for hydrolysis, heating, centrifuging, collecting supernatant, drying, and dissolving with 500 μl acetonitrile/water solution.

The concentration of the fructosidase solution in the step (2) of the invention is as follows: 50-200U/mL.

Preferably, the concentration of the fructosidase solution in step (2) of the present invention is: 100U/mL.

The heating in the step (2) of the invention is as follows: heated at 80℃for 20 minutes.

The setting conditions of the oscillator in the step (2) of the invention are as follows: the temperature is 55 ℃, the rotating speed is 200 revolutions per minute, and the time is 1-7 hours.

Preferably, the setting conditions of the oscillator in the step (2) of the present invention are: the temperature was 55℃and the rotational speed was 200 revolutions per minute for 3 hours.

The setting conditions of the centrifugation in the step (2) of the invention are as follows: 4500r/min,15min.

The acetonitrile/water solution in the step (2) is as follows: the volume ratio is 1:1.

Compared with the prior art, the invention has the following beneficial effects:

1. according to the invention, the polygonatum polysaccharide is hydrolyzed by adopting an enzymolysis method, fructosidase suitable for polygonatum is screened out, and enzymolysis conditions and methods are optimized, so that when the enzyme concentration is 100U/mL, more chromatographic peaks of oligosaccharides with higher polymerization degree are obtained; at a hydrolysis time of 3 hours, the number and area of peaks are relatively best; the fingerprint spectrum of the final rhizoma polygonati polysaccharide can reach 17 chromatographic peaks, and the saccharide components with the polymerization degree of 7-8 can be detected, so that the high-efficiency and accurate research system plays an important role in further promoting research application and rapid development of the rhizoma polygonati polysaccharide.

2. The specific oligosaccharide fragments in the polygonatum polysaccharide are separated and detected by a glycosidase enzymolysis method, the enzymatic hydrolysis condition is mild, the specificity is strong, the hydrolysis reaction has more directionality, purpose and controllability, and the method has remarkable advantages in the aspects of analyzing the glycosidic bond type of the polysaccharide, constructing a sugar spectrum, researching the structure-activity relationship and the like; solves the problems that partial acid hydrolysis products in the phenol-sulfuric acid method commonly adopted in the prior art are generally complex, poor in uniformity, low in accuracy, incapable of obtaining structural information of polysaccharide, incapable of effectively evaluating the quality of rhizoma polygonati polysaccharide from different sources and the like.

3. The invention adopts a combination method of a sensitive evaporative light detector and an HILIC chromatographic column, can separate and detect oligosaccharides without carrying out pre-column derivatization, and the result shows that all peaks are separated, the peak property is good, the resolution is high, the specificity is good, and the problem of poor specificity of the traditional rhizoma polygonati quality detection method is solved.

4. The invention carries out methodology investigation on the established fingerprint spectrum, and results are: (1) Through a precision test, the RSD of the relative retention time and the relative peak area of the common peak is measured to be less than 3%, which indicates that the precision of the instrument is good and meets the fingerprint spectrum requirement; (2) The relative retention time of each common peak and the RSD of the relative peak area are measured to be less than 3% through a repeatability test, and the results of each extraction of the same sample are consistent, so that the extraction method has good repeatability and meets the fingerprint spectrum requirement; (3) Through a stability test, the relative retention time of the common peak and the RSD of the relative peak area are measured to be less than 3%, which shows that the sample solution is stable within 24 hours, the chemical components and the content are not changed, and the fingerprint spectrum requirements are met; in conclusion, the method of the invention is stable and feasible.

5. According to the method, through fingerprint similarity evaluation, 17 peaks corresponding to enzyme hydrolysis products are detected through HPLC-HILIC-ELSD, and DPs with peaks of 1-7 are preliminarily determined through comparison of retention time of fructose, glucose and sucrose under the same conditions; 5 batches of rhizoma polygonati, 5 batches of rhizoma polygonati yunnanensis, 10 batches of polygonatum cyrtonema, 5 batches of Sichuan rhizoma polygonati, 5 batches of Hubei rhizoma polygonati, 2 batches of small leaf rhizoma polygonati and 2 batches of imported rhizoma polygonati medicinal material chromatograms are respectively imported into a traditional Chinese medicine fingerprint similarity evaluation system (2.0 edition) software to generate control maps of 3 basic raw rhizoma polygonati, 34 batches of samples are respectively compared with the control maps of rhizoma polygonati, rhizoma polygonati yunnanensis and polygonatum cyrtonema, and similarity is calculated as a result:

(1) and (3) withPolygonatum cyrtonema Fall et Sieb3 primordia compared to control profileAmong the similarity of the rhizoma polygonati, the similarity of 10 batches of polygonatum cyrtonema is more than 0.93. Compared with Polygonatum kingianum and Polygonatum sibiricum, the similarity is less than 0.85 except for 1 sample of Polygonatum kingianum (D1). Compared with the comparison map of the polygonatum cyrtonema, the easily mixed products in the market are distributed more discretely, wherein the similarity of the polygonatum cyrtonema in Hubei is between 0.44 and 0.95; except for 1 lot (C4), the similarity of Sichuan rhizoma polygonati is less than 0.86; the similarity of the imported rhizoma polygonati is less than 0.82; the similarity of the rhizoma polygonati of small leaves is less than 0.5;

(2) and (3) withPolygonatum kingianum (Willd.) DrAnd comparing the control patterns, wherein the similarity of 5 batches of Polygonatum kingianum medicinal materials in the similarity of 3 primitive Polygonatum kingianum is more than 0.90, and the similarity of the Polygonatum kingianum, the Polygonatum cyrtonema and the Polygonatum cyrtonema is less than 0.90 except for the sample of Polygonatum cyrtonema (S1, S8, S9 and S10) and the sample of Polygonatum sibiricum (J2). Compared with other commercially available rhizoma polygonati, the similarity is 0.65-0.99, wherein the similarity of Sichuan rhizoma polygonati is more than 0.90; the similarity between the Hubei rhizoma Polygonati and the imported rhizoma Polygonati is more than 0.90; the ratio of rhizoma polygonati is less than 0.80.

(3) And (3) withRhizoma polygonati of chicken headComparing the comparison patterns, wherein the similarity of 5 batches of rhizoma polygonati with the number of 5 of rhizoma polygonati with the number of 3 primordial groups is more than 0.99, and the similarity of rhizoma polygonati with the number of flowers is less than 0.80 except for samples of rhizoma polygonati with the number of flowers (S7) and rhizoma polygonati with the number of flowers (D4). Compared with other commercially available rhizoma polygonati, the similarity is 0.60-0.99, wherein the similarity of Sichuan rhizoma polygonati is less than 0.80; the similarity of the polygonatum sibiricum in Hubei except H5 is less than 0.70; the similarity between the rhizoma polygonati import and the rhizoma polygonati lobae is more than 0.90 except the rhizoma polygonati import (Z2).

The results show that the intraspecific fingerprint chromatographic similarity of the three rhizoma polygonati medicinal materials from different sources recorded in the pharmacopoeia is higher, and the difference is smaller; the difference among the species of the 3-species original rhizoma polygonati is large; some easy-to-mix products exist in the market and the rhizoma polygonati species in the three parts recorded in the pharmacopoeia also have a little difference. Therefore, the different polygonatum polysaccharides have different structural characteristics, and are convenient to distinguish.

6. The invention has the advantages of but is not limited to the research on the variety of the rhizoma polygonati recorded in pharmacopoeia in terms of sample collection, and solves the problem of identifying the rhizoma polygonati miscible products such as Sichuan rhizoma polygonati, hubei rhizoma polygonati and the like in the current market to a certain extent.

7. The invention analyzes the chemometry of the polygonatum sibiricum varieties recorded in pharmacopoeia:

(1) And (3) cluster analysis: the 20 batches of rhizoma Polygonati samples were divided into 3 major classes: class 1 includes 10 batches of Polygonatum cyrtonema, respectively Anhui Jinzhai, shitai (3), zhejiang Hangzhou (2), chun an (2), guangzhou, jiangxi Ruichang; class 2 includes 5 batches of Polygonatum kingianum, sichuan Dada, chengdu (2), hubei Wuhan, yunnan Kunming, and Guizhou Zhuangjin, respectively; class 3 includes 5 batches of rhizoma Polygonati, each of Guizhou Dafang, anhui Jizhou, shandong Zaozhuang and Liaoning Qing Yuan;

(2) And (3) principal component analysis: 17 common peak areas of 20 batches of samples are imported into SPSS 23.0 software for PCA analysis, and feature values are obtained>1 is an extraction standard, 4 main components are extracted, the first three PCs respectively account for 50.1%, 26.6% and 9.1% of the total variance, and 3 main components represent 85.8% of the information content of 17 component amounts in the polygonatum polysaccharide, so that the quality of the polygonatum polysaccharide is sufficiently evaluated; introducing the data matrix into SIMCA statistical software, extracting 3 principal components, R in the model ² X(cum)＝0.844，Q ² (cum) =0.612, all greater than 0.5, indicating that the better the predictive power of the PCA model. The three-dimensional PCA score plot of PCP shows that 20 PCPs were classified into 3 classes, consistent with the HCA results; polygonatum cyrtonema S1-S10 is gathered into one group, polygonatum cyrtonema S11-S15 is gathered into another group, and the rhizoma polygonati of chicken head S16-S20 are divided into a third group;

(3) Partial least squares discriminant analysis: the data matrix was imported into SIMCA statistical software for analysis by PLS-DA, R in this model ² X(cum)＝0.909，R ² Y(cum)＝0.863，Q ² (cum)＝0.775，R ² Y and Q ² Preferably greater than 0.5, and R ² Y and Q ² The closer to 1 the value of (c) is, the better the predictive power of the PLS-DA model is. The analysis results are shown in FIG. 12, and the classification results are consistent with HCA and PCA. FIG. 13 reflects the VIP value of the model, the magnitude of the VIP value representing the magnitude of each argument contribution, in VIP>1 is a screening standard to obtain a marker component which causes the difference between the production areas; from the VIP values, VIP values of chromatographic peak 1, chromatographic peak 4, chromatographic peak 5, chromatographic peak 7 and chromatographic peak 8 are all greater than 1; the three analysis methods verify that the analysis of the polygonatum polysaccharide by adopting the enzymolysis method is stable and accurateIs feasible.

Drawings

FIG. 1 is a chromatogram showing the effect of enzyme concentration of 10U/mL on PCP hydrolysis;

FIG. 2 is a chromatogram showing the effect of enzyme concentration of 50U/mL on PCP hydrolysis;

FIG. 3 is a chromatogram showing the effect of enzyme concentration of 100U/mL on PCP hydrolysis;

FIG. 4 is a chromatogram showing the effect of enzyme concentration of 200U/mL on PCP hydrolysis;

FIG. 5 is a chromatogram showing the effect of a reaction time of 1 hour on PCP hydrolysis;

FIG. 6 is a chromatogram showing the effect of a reaction time of 3 hours on PCP hydrolysis;

FIG. 7 is a chromatogram showing the effect of a reaction time of 5 hours on PCP hydrolysis;

FIG. 8 is a chromatogram showing the effect of a reaction time of 7 hours on PCP hydrolysis;

FIG. 9 is a chromatogram showing the effect of 5mg samples on PCP hydrolysis;

FIG. 10 is a chromatogram showing the effect of 10mg samples on PCP hydrolysis;

FIG. 11 is a chart showing the separation effect of a chromatographic column Sunniest HILIC-S;

FIG. 12 is a diagram showing the effect of chromatographic column Welch Ultimate HILIC Amide on separation

FIG. 13 shows a chromatographic column Hypersil GOLD ^TM PEI HILIC HPLC separation effect diagram

FIG. 14 is a chromatogram showing the effect of column temperature 30℃on PCP enzymatic hydrolysis;

FIG. 15 is a chromatogram showing the effect of column temperature 35℃on PCP enzymatic hydrolysis;

FIG. 16 is a chromatogram showing the effect of column temperature 40℃on PCP enzymatic hydrolysis;

FIG. 17 is a chromatogram showing the separation effect of mobile phase methanol-water on a sample;

FIG. 18 is a chromatogram showing the separation effect of mobile phase acetonitrile-water on a sample;

FIG. 19 is a chromatogram showing the effect of evaporation light drift tube temperature 90℃on PCP enzymatic hydrolysis;

FIG. 20 is a chromatogram showing the effect of evaporation light drift tube temperature 100℃on PCP enzymatic hydrolysis;

FIG. 21 is a chromatogram showing the effect of evaporation light drift tube temperature 105℃on PCP enzymatic hydrolysis;

FIG. 22 is a chromatogram showing the effect of 5. Mu.L of sample loading on PCP hydrolysis;

FIG. 23 is a chromatogram showing the effect of 10. Mu.L of sample loading on PCP hydrolysis;

FIG. 24 is a chromatogram showing the effect of 15. Mu.L of sample loading on PCP hydrolysis;

FIG. 25 is a chromatogram of a sample of Polygonatum sibiricum and a control;

FIG. 26 is a chromatogram of a fructose control;

FIG. 27 is a chromatogram of a glucose control;

FIG. 28 is a chromatogram of a sucrose control;

FIG. 29 is a fingerprint of 10 batches of Polygonatum cyrtonema Fabricius; (S1-S10 is Polygonatum cyrtonema Sieb. Et Zucc. Of Polygonatum cyrtonema Sieb

FIG. 30 is a fingerprint chart of rhizoma Polygonati Odorati, and rhizoma Polygonati Odorati; ( D1-D5 are Polygonatum kingianum; J1-J5 are rhizoma polygonati; H1-H5 is Polygonatum sibiricum of Hubei province )

FIG. 31 is a fingerprint chart of rhizoma Polygonati Odorati, sichuan rhizoma Polygonati and imported rhizoma Polygonati; ( C1-C5 is Sichuan rhizoma Polygonati; Z1-Z2 are imported rhizoma Polygonati; X1-X2 is Polygonatum sibiricum Red )

FIG. 32 is a graph showing HCA tree relationship among 20 batches of Polygonatum sibiricum samples; ( Wherein 1-10 is Polygonatum cyrtonema Fabricius; 11-15 parts of Polygonatum kingianum; 16-20 chicken head sealwort )

FIG. 33 is a lithotripsy of 20 batches of Polygonatum sample;

FIG. 34 is a three-dimensional projection view of principal component analysis of 20 batches of rhizoma Polygonati samples;

FIG. 35 is a partial least squares discriminant analysis of 20 batches of Polygonatum sibiricum samples;

fig. 36 is a VIP plot of 20 batches of rhizoma Polygonati samples.

The technical scheme of the invention is further specifically described by the following specific examples.

Example 1 method for establishing a multiple fingerprint of a Polygonatum sibiricum original species

(1) Preparation of a control solution: taking 10mg of fructose, sucrose and glucose reference substances respectively, precisely weighing, transferring into a10 mL volumetric flask, and fixing the volume of water to a scale to obtain the final product;

(2) Preparation of test solution: adding 10mL of water into 5mg of rhizoma polygonati polysaccharide to prepare a rhizoma polygonati polysaccharide solution with the concentration of 0.5mg/mL, and then mixing 2.5mL of rhizoma polygonati polysaccharide solution with 2.5mL of fructosidase solution (100U/mL); the mixture was hydrolyzed in a shaker (55 ℃,200 rpm, 3 hours) and heated at 80 ℃ for 20 minutes to denature the enzyme; centrifuging (4500 r/min,15 min), collecting supernatant, drying, and dissolving with 500 μl acetonitrile/water (1:1, v/v) solution;

gradient elution conditions: 0-25 min, 85-65% of A; 25-35 min, 65-50% of A.

Example 2 method for establishing a Multi-element fingerprint of a Polygonatum sibiricum Red original species

(1) Preparation of a control solution: taking 10mg of fructose, sucrose and glucose reference substances, precisely weighing, transferring into a10 mL volumetric flask, and fixing the volume of water to a scale to obtain the product;

(2) Preparation of test solution: 2.5mL of the Polygonatum sibiricum polysaccharide solution prepared in example 1 was mixed with 2.5mL of the fructosidase solution (50U/mL); the mixture was hydrolyzed in a shaker (55 ℃,200 rpm, 1 hour) and heated at 80 ℃ for 20 minutes to denature the enzyme; centrifuging (4500 r/min,15 min), collecting supernatant, drying, and dissolving with 500 μl acetonitrile/water (1:1, v/v) solution;

chromatographic column: hypersil GOLD ^TM PEI HILIC HPLC, model is: 5 μm,250 mm. Times.4.6 mm; mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradientEluting; column temperature: 35 ℃; flow rate: 1.0 mL/min ^-1 The method comprises the steps of carrying out a first treatment on the surface of the A detector: detecting evaporation light; the sample injection amount is 10 mu L;

gradient elution conditions: 0-25 min, 85-65% of A; 25-35 min, 65-50% of A.

Example 3 method for establishing a Multi-element fingerprint of a Polygonatum sibiricum Red original species

(2) Preparation of test solution: 2.5mL of the Polygonatum sibiricum polysaccharide solution prepared in example 1 was mixed with 2.5mL of the fructosidase solution (200U/mL); the mixture was hydrolyzed in a shaker (55 ℃,200 rpm, 7 hours) and heated at 80 ℃ for 20 minutes to denature the enzyme; centrifuging (4500 r/min,15 min), collecting supernatant, drying, and dissolving with 500 μl acetonitrile/water (1:1, v/v) solution;

gradient elution conditions: 0-25 min, 85-65% of A; 25-35 min, 65-50% of A.

Example 4 method for establishing a multiple fingerprint of a Polygonatum sibiricum original species

(2) Preparation of test solution: 2.5mL of the Polygonatum sibiricum polysaccharide solution prepared in example 1 was mixed with 2.5mL of the fructosidase solution (100U/mL); the mixture was hydrolyzed in a shaker (55 ℃,200 rpm, 5 hours) and heated at 80 ℃ for 20 minutes to denature the enzyme; centrifuging (4500 r/min,15 min), collecting supernatant, drying, and dissolving with 500 μl acetonitrile/water (1:1, v/v) solution;

gradient elution conditions: 0-25 min, 85-65% of A; 25-35 min, 65-50% of A.

To further verify the feasibility of the invention, the inventors performed a series of experiments, specifically as follows:

1. fingerprint establishment method and fumbling test

1 instrument and reagent

1.1 instruments

U3000 high performance liquid chromatograph (equipped with AllechELSD6000 evaporative light scattering detector Daian);

ML204 parts per million electronic balance, XP26 parts per million electronic balance, toli multi instruments Shanghai limited;

elmasonic S30 (H) ultrasonic cleaner, deluxe technologies inc;

advantage A10 Milli-Q ultra pure water instrument, mirui biosciences Inc.

1.2 reagents

The samples were 34 batches in total, and are market blendable samples, and specific source specifications are shown in table 1.

TABLE 1 detailed listing of test sample materials

Glycosidase: sucrase (Shanghai-derived leaf organism); the specification of the fructosidase is 100U/Mg.

Control and other reagents: fructose control (lot 111504-201703, purity 99.8%, provided by the national food and drug verification institute), sucrose control (lot 111507-201303, purity 99.8%, provided by the national food and drug verification institute); glucose control (Sigma). The acetonitrile in the reagents used in the experiment is chromatographic purity, and the rest are analytical purity; the water is purified water.

2. Experimental method

2.1 Examination of sample preparation method

The research pre-establishes a common chromatographic condition of the rhizoma polygonati with different sources under the hydrolysis of different glycosidases, namely that under the condition, main chromatographic peaks can be well separated after the hydrolysis of the different glycosidases.

2.1.1 investigation of glycosidase concentration

Diluting fructosidase with water to obtain enzyme solution with proper concentration, namely 10U/mL, 50U/mL,

100U/mL and 200U/mL, wherein:

10U/ml: to fix the volume to 10ml, 100U of enzyme is needed, namely 1mg of fructosidase is taken;

50U/ml: to fix the volume to 10ml, 500U of enzyme is needed, namely 5mg of fructosidase is taken;

100U/ml: to fix the volume to 10ml, 1000U of enzyme is needed, namely 10mg of fructosidase is taken;

200U/ml: to determine the volume to 10ml, 2000U of enzyme was required, i.e., 20mg of fructosidase was taken.

The fructosidase is diluted with water to a suitable concentration of enzyme solution, i.e., 10U/mL, 50U/mL, 100U/mL, and 200U/mL. 5mg of PCPs were accurately weighed into a10 mL centrifuge tube, four parts were parallel, and then mixed with different enzyme solutions (2.5 mL). The mixture was shaken 3 times (55 ℃ C., 200r/min,3 h) in a shaker to carry out the hydrolysis. The mixture was then heated at 80℃for 20 minutes to denature the enzyme. After centrifugation (4500 r/min,15 min), the supernatant was taken, dried, dissolved in 500. Mu.L acetonitrile/water (1:1, v/v) solution and further tested in HILIC mode.

The effect of different enzyme concentrations on hydrolysis is shown in figures 1-4: at an enzyme concentration of 10U/mL, there was little chromatographic peak for the oligosaccharides. The response of the oligosaccharide peak was very low at an enzyme concentration of 50U/mL. When the enzyme concentration is 100U/mL, the chromatographic peak of the oligosaccharide having a higher polymerization degree is more than that of the oligosaccharide having an enzyme concentration of 200U/mL. To obtain more oligosaccharide information, 100U/mL was chosen as the optimal enzyme concentration.

2.1.2 investigation of enzymatic hydrolysis method

PCPs solution (5 mg) was mixed with fructosidase (100U/mL, 2.5 mL) in four portions in parallel. The mixture was shaken in a shaker (55 ℃,200 r/min) for 1h, 3h, 5h and 7h, respectively. The mixture was then heated at 80℃for 20 minutes to denature the enzyme. After centrifugation (4500 r/min,15 min), the supernatant was taken, dried and dissolved in 500. Mu.L of acetonitrile, water (1:1, v/v) solution.

The effect of reaction time on hydrolysis is shown in FIGS. 5-8. As the reaction time increases, the higher degree of polymerization oligosaccharides are further hydrolyzed to lower degree of polymerization oligosaccharides. The two chromatographic peaks with retention times of 15-25 min in the hydrolyzate 3h chromatogram increased in area, while the chromatographic peaks with retention times of 27-40 min increased compared to hydrolyzate 1 h. However, as the hydrolysis time continued to increase, the number and area of peaks did not change significantly. Therefore, 3 hours was selected as the optimal hydrolysis time in consideration of the hydrolysis efficiency and effect.

2.1.3 investigation of sample size

Relates to the mixing problem of polysaccharide solution and glycosidase solution, and the final volume of the sample to be tested is set to be 2mL. The sample weights of the samples of 5mg and 10mg were examined, and the results were compared with those of FIGS. 9 to 10, and it was found that the number and area of peaks did not significantly change as the sample weights continued to increase. Therefore, 10mg was selected as the optimal sampling amount in consideration of the experimental efficiency and effect.

2.1.4 determination of the preparation method of the sample

Sample solution preparation: 5mg of PCP was mixed with 2.5mL of a fructosidase solution (100U/mL). The mixture was hydrolyzed in a shaker (55 ℃,200 rpm, 3 hours) and heated at 80 ℃ for 20 minutes to denature the enzyme. After centrifugation (4500 r/min,15 min), the supernatant was taken, dried, and dissolved in 500. Mu.L acetonitrile/water (1:1, v/v) solution for HILIC-HPLC analysis.

2.1.5 preparation of control solution

And (3) taking about 10mg of fructose, sucrose and glucose, precisely weighing, transferring to a10 mL volumetric flask, and fixing the volume of water to a scale.

2.2 optimization of chromatographic conditions

2.2.1 selection of detectors

Compared with differential detection (HPLC-RID), the evaporative light detection (HPLC-ELSD) has the advantages of stable baseline, good separation effect, high response value, accurate result, good reproducibility and the like, and the pretreatment for polysaccharide detection is relatively simple, oligosaccharide can be separated and detected without pre-column derivatization, and the result shows that all peaks are separated, the peak shape is good, and the resolution is high.

2.2.2 selection of chromatographic columns

In this study, a series of HILIC columns (Sunniest HILIC-S, welch Ultimate HILIC Amide and Hypersil GOLD were studied ^TM PEI HILIC HPLC) possibility of separating the polysaccharide hydrolysate of Polygonatum sibiricum Red. We found Hypersil GOLD ^TM PEI HILIC HPLC is suitable for separating rhizoma Polygonati polysaccharide hydrolysate, and has good separation resolution and peak distribution. See fig. 11-13.

2.2.3 investigation of column temperature

The results of studies on the degree of separation and peak shape of the same sample at a set column temperature of 30, 35 and 40 show that the temperature has little influence on the separation and peak shape of chromatographic peaks as shown in FIGS. 14 to 16, and the degree of separation at a set column temperature of 35 is slightly better than those at 30 and 40 ℃. Therefore, the column temperature was selected to be 35 ℃.

2.2.4 investigation of mobile phases and elution gradients

The research successively examines the HPLC chromatographic peak separation effects of different samples under the mobile phase of acetonitrile-water, methanol-water and the like, and compares and discovers that the separation effects of different rehmannia root refined products of the acetonitrile-water system are optimal. In order to realize the separation of the main chromatographic peaks of different radix rehmanniae concentrates, the elution gradient is further optimized in the experiment, and finally the linear elution mode is determined, as shown in table 2. See fig. 17-18.

TABLE 2 gradient elution System for mobile phases

2.2.5 investigation of the temperature of the Evaporation light drift tube

The study examines different drift tube temperatures at 90 ℃, 100 ℃ and 105 ℃ and the experimental result shows that the different drift tube temperatures have larger influence on the base line. As shown in fig. 19 to 21, the base line is stable when the temperature of the drift tube is 100 ℃. The experiment selects the temperature of the drift tube to be 100 ℃.

2.2.6 investigation of sample injection amount

In the research, the chromatographic separation effect and the peak information quantity of different sample injection amounts of 5 mu L, 10 mu L and 15 mu L of the same batch of samples are examined, and as shown by comparing the figures 22-24, the chromatographic peak area is smaller when the sample injection amount is 5 mu L, and the chromatographic peak with longer retention time is easily ignored, so that the subsequent further analysis of the oligosaccharide fragments is not facilitated; the sample injection amount is 15 mu L, and the chromatographic peak line of the partial sample is poor. The sample injection amount of the study was considered to be 10. Mu.L.

2.2.7 determination of chromatographic conditions

Chromatographic column: hypersil GOLD ^TM PEI HILIC HPLC(5μm，250mm×4.6mm)

Mobile phase: acetonitrile (a), water (B);

elution procedure: 0-25 min, 85-65% of A; 25-35 min, 65-50% of A;

a detector: evaporation light detector

Flow rate: 1.0 mL/min ^-1 ；

Column temperature: 35 ℃; sample injection amount 10. Mu.L

Under the above conditions, rhizoma Polygonati of different sources can be well separated from other impurity peaks, and the separation degree of the rhizoma Polygonati from adjacent peaks is greater than 1.5, and the graphs of the reference substances and the samples are shown in fig. 25-28.

2.3 methodology investigation

2.3.1 precision test

Taking one part of the S2 test sample, preparing a test sample solution according to the content of 2.1, continuously injecting sample for 6 times, and recording the relative retention time and the relative peak area of each common peak (taking the peak outlet time and the peak area of fructose as reference substances, and the ratio of the peak outlet time and the peak area of other peaks to the peak outlet time and the peak area of the other peaks, which are the same below), wherein the RSD of the measured common peak relative retention time and the RSD of the measured common peak relative peak area are less than 3%, which indicates that the instrument precision is good and the fingerprint spectrum requirements are met.

2.3.2 repeatability experiments

Taking 6 parts of an S2 test sample, preparing a test sample solution according to a method under a term "2.1", and measuring according to a chromatographic condition under a term "2.1", wherein the relative retention time of each common peak and the RSD of the relative peak area are measured to be less than 3%, and the results of each extraction of the same sample are consistent, so that the extraction method has good reproducibility and meets the fingerprint spectrum requirement.

2.3.3 stability experiments

Taking the same part of S2 sample solution, and respectively sampling at 0, 2, 4, 8, 12 and 24 hours, wherein the measured relative retention time of the common peak and the RSD of the relative peak area are less than 3%, which indicates that the sample solution is stable within 24 hours, the chemical components and the content of the sample solution are not changed, and the sample solution meets the fingerprint requirements.

3. Chemometric analysis

3.1.1 hierarchical clustering analysis

The common peak areas of HILIC-HPLC-ELSD fingerprint of rhizoma Polygonati polysaccharide recorded in 20 batches of pharmacopoeias are integrated to form a 20×17 data matrix. The data matrix is subjected to low-level fusion and imported into the SPSS 23, and an average correlation method and Euclidean distance are adopted as a clustering method and a similarity measure. The samples are classified according to individual differences.

3.1.2 principal component analysis

The 20×17 data matrix is imported into the SPSS 23, and the feature value and the variance accumulation contribution rate are obtained by selecting a dimension reduction method. And further selecting a variable with a characteristic value of >1 for principal component analysis. The 20 x 17 data matrix was then imported into SIMCA 13 statistical software for PCA analysis.

3.1.3 partial least squares discriminant analysis

The 20X 17 data matrix was imported into SIMCA 13 statistical software for PLS-DA analysis. The effect of each chromatographic peak on the discrimination results is determined by VIP (variable importance in projection) values.

Results 4 results

The research collects 5 batches of rhizoma polygonati of chicken head, 5 batches of rhizoma polygonati of Yunnan, 10 batches of polygonatum cyrtonema, 5 batches of Sichuan rhizoma polygonati, 5 batches of Hubei rhizoma polygonati, 2 batches of small leaf rhizoma polygonati and 2 batches of imported rhizoma polygonati, and 34 batches of samples are subjected to HPLC-ELSD analysis research to find out whether the differences of three different basic raw rhizoma polygonati exist in pharmacopoeia and whether the differences of the three basic rhizoma polygonati and the easy-to-mix product exist in the market or not so as to identify.

4.1 evaluation of finger print similarity

As shown in FIGS. 29-31, 17 peaks were detected by HPLC-HILIC-ELSD corresponding to the enzyme hydrolysate. DPs with peaks ranging from 1 to 7 were initially determined by comparing the retention times of fructose, glucose and sucrose under the same conditions as shown in FIG. 7. And respectively introducing the chromatograms of 5 batches of rhizoma polygonati, 5 batches of rhizoma polygonati yunnanensis, 10 batches of polygonatum cyrtonema, 5 batches of Sichuan rhizoma polygonati, 5 batches of Hubei rhizoma polygonati, 2 batches of small-leaf rhizoma polygonati and 2 batches of imported rhizoma polygonati into a traditional Chinese medicine fingerprint similarity evaluation system (version 2.0) software to generate control maps of 3 basic rhizoma polygonati. And comparing 34 batches of samples with control patterns of rhizoma polygonati, rhizoma polygonati yunnanensis and rhizoma polygonati cyrtomii, and calculating the similarity, wherein the results are shown in tables 3-5. As can be seen from the figures:

(1) compared with the control pattern of Polygonatum cyrtonema Fabricius, the similarity of 10 batches of Polygonatum cyrtonema Fabricius among the similarity of 3 primitive Polygonatum cyrtonema Fabricius is more than 0.93. Compared with Polygonatum kingianum and Polygonatum sibiricum, the similarity is less than 0.85 except for 1 sample of Polygonatum kingianum (D1). Compared with the comparison map of the polygonatum cyrtonema, the easily mixed products in the market are distributed more discretely, wherein the similarity of the polygonatum cyrtonema in Hubei is between 0.44 and 0.95; except for 1 lot (C4), the similarity of Sichuan rhizoma polygonati is less than 0.86; the similarity of the imported rhizoma polygonati is less than 0.82; the similarity of the rhizoma polygonati of small leaves is less than 0.5;

(2) compared with the Polygonatum kingianum control spectrum, the similarity of 5 batches of Polygonatum kingianum medicinal materials in the similarity of 3 primitive Polygonatum kingianum is more than 0.90, and the similarity of the Polygonatum kingianum and the Polygonatum cyrtonema is less than 0.90 except for the sample of Polygonatum cyrtonema (S1, S8, S9, S10) and Polygonatum sibiricum (J2). Compared with other commercially available rhizoma polygonati, the similarity is 0.65-0.99, wherein the similarity of Sichuan rhizoma polygonati is more than 0.90; the similarity between the Hubei rhizoma Polygonati and the imported rhizoma Polygonati is more than 0.90; the ratio of rhizoma polygonati is less than 0.80.

(3) Compared with the comparison map of the rhizoma polygonati, the similarity of 5 batches of rhizoma polygonati with the 3 types of basic groups is more than 0.99, and the similarity of the rhizoma polygonati with the flos polygonati is less than 0.80 except for the sample of the rhizoma polygonati with the flos polygonati (S7) and the sample of the rhizoma polygonati with the flos polygonati. Compared with other commercially available rhizoma polygonati, the similarity is 0.60-0.99, wherein the similarity of Sichuan rhizoma polygonati is less than 0.80; the similarity of the polygonatum sibiricum in Hubei except H5 is less than 0.70; the similarity between the rhizoma polygonati import and the rhizoma polygonati lobae is more than 0.90 except the rhizoma polygonati import (Z2).

TABLE 3 similarity evaluation results

Note that: the reference spectrum is a Polygonatum cyrtonema Fabricius reference spectrum (R1) chromatogram, and the reference spectrum is generated by performing multipoint correction by using a median method. The similarity of each batch was compared to R1. ( S1-S10 polygonatum cyrtonema; J1-J5 rhizoma polygonati; D1-D5 Polygonatum kingianum; C1-C5 Sichuan rhizoma polygonati; H1-H5 Polygonatum sibiricum Red; Z1-Z2 Polygonatum sibiricum; X1-X2 import Polygonatum sibiricum Red )

TABLE 4 similarity evaluation results

Note that: the control spectrum (R2) is obtained by taking the Polygonatum kingianum control spectrum as a reference spectrum and performing multi-point correction by using a median method. The similarity of each batch was compared to R2.

TABLE 5 similarity evaluation results

Note that: the control spectrum (R3) is used as a reference spectrum, and the control spectrum is generated by performing multipoint correction by using a median method. The similarity of each batch was compared to R3.

4.2 Cluster analysis

And selecting three rhizoma polygonati varieties (20 batches in total) recorded in pharmacopoeia, and sorting the areas of the common peaks and peaks in the chromatogram of the rhizoma polygonati polysaccharide sample enzyme hydrolysis product to obtain a 20 multiplied by 17 data matrix, introducing IBM SPSS Statistic edition software, and adopting inter-group connection as a clustering method. As shown in fig. 32, 20 batches of rhizoma Polygonati samples were classified into 3 major categories: class 1 includes 10 batches of Polygonatum cyrtonema, respectively Anhui Jinzhai, shitai (3), zhejiangzhou (2), linqi (2), quzhou, jiangxi Ruichang; class 2 includes 5 batches of Polygonatum kingianum, sichuan Dada, chengdu (2), hubei Wuhan, yunnan Kunming, and Guizhou Zhuangjin, respectively; class 3 includes 5 batches of rhizoma Polygonati, each of Guizhou Dafang, anhui Jizhou, shandong Zaozhuang and Liaoning Qing Yuan. The results show that the polygonatum polysaccharase hydrolysate is sequentially divided into three categories with a certain correlation with the basic source after data analysis, and the same category in the sample subjected to cluster analysis shows that the polygonatum polysaccharase hydrolysate has high similarity in structural characteristics. The reason for the division into 3 types is probably caused by the fact that the structures of the polygonatum polysaccharide from different sources have certain differences, and the discovery lays a foundation for the identification work of the polygonatum.

4.3 principal component analysis

The principal component analysis embodying the dimension reduction idea is to select components with larger contribution rate for analysis, so that the analysis process is simplified, and original information is displayed as much as possible. 17 common peak areas of 20 batches of samples were imported into SPSS 23.0 software for PCA analysis, the eigenvalues and variance contribution ratios of the principal component analysis are shown in Table 4, and the lithotriptic map is shown in FIG. 33. By characteristic value>1 is the extraction standard, 4 main components are extracted, and the first three PCs respectively account for 50.1 percent of the total variance26.6% and 9.1%,3 principal components were selected for evaluation, which represents an information amount of 85.8% of 17 component amounts in the polygonatum polysaccharide, sufficient for evaluating the quality of the polygonatum polysaccharide. Introducing the data matrix into SIMCA statistical software, extracting 3 principal components, R in the model ² X(cum)＝0.844，Q ² (cum) =0.612, all greater than 0.5, indicating that the better the predictive power of the PCA model. The three-dimensional PCA score plot of PCPs (fig. 34) shows that 20 PCPs were classified into 3 classes, consistent with HCA results. Polygonatum cyrtonema S1-S10 are clustered into a group. Polygonatum kingianum S11-S15 is clustered into another group, and Polygonatum kingianum S16-S20 is clustered into a third group. The PCA model also provides satisfactory classification for the rhizoma polygonati of three different origins, which shows that the rhizoma polygonati polysaccharide can be well identified by analyzing the rhizoma polygonati polysaccharide by adopting an enzyme hydrolysis method.

TABLE 4 eigenvalue and variance contribution rate total variance interpretation

2.5.3 partial least squares discriminant analysis

PLS-DA is commonly used for discriminant analysis of samples of a multivariate system, and is suitable for situations where collinearity exists between independent variables or where the number of samples is small. The data matrix was imported into SIMCA statistical software for analysis by PLS-DA, R in this model ² X(cum)＝0.909，R ² Y(cum)＝0.863，Q ² (cum)＝0.775，R ² Y and Q ² Preferably greater than 0.5, and R ² Y and Q ² The closer to 1 the value of (c) is, the better the predictive power of the PLS-DA model is. The analysis results are shown in FIG. 35, and the classification results are consistent with HCA and PCA. FIG. 36 reflects the VIP value of the model, the magnitude of the VIP value representing the magnitude of each argument contribution, in VIP>1 is a screening criteria to derive a marker component that causes inter-producing-area differences. From the VIP value, the colorVIP values for spectral peak 1, chromatographic peak 4, chromatographic peak 5, chromatographic peak 7 and chromatographic peak 8 are all greater than 1. The above results demonstrate that the above 5 chromatographic peaks have a large contribution to the discriminant analysis of the PLS-DA model.

5. Conclusion(s)

Through research, the present market has disordered rhizoma polygonati base, particularly, the rhizoma polygonati with multiple flowers and the rhizoma polygonati with chicken heads are difficult to identify to a certain extent in three types of rhizoma polygonati recorded in pharmacopoeia, and rhizoma polygonati medicinal materials with cultivation variation and imported rhizoma polygonati medicinal materials are also present, and the rhizoma polygonati with cultivation variation is different from the conventionally used rhizoma polygonati in shape to a certain extent, but accords with the characteristics described in pharmacopoeia, so that the rhizoma polygonati with cultivation variation is difficult to distinguish; imported rhizoma polygonati mostly enters the Chinese market from the countries such as Burmese and Vietnam, and the imported rhizoma polygonati is large in size, mostly serves as the rhizoma polygonati to be sold, and is high in content, but the difference between the imported rhizoma polygonati and the common rhizoma polygonati varieties in China in efficacy and components is unknown. In addition, in recent years, a large number of non-pharmacopoeia rhizoma polygonati called evergreen rhizoma polygonati is planted in places such as Sichuan of Guizhou, and the like, because the rhizoma polygonati is fast in asexual propagation, the evergreen is easy to field management and high in yield, the rhizoma polygonati has a trend of increasing planting area, and although the rhizoma polygonati medicinal material is not obviously different from the pharmacopoeia in appearance, the rhizoma polygonati medicinal material is not a pharmacopoeia genuine product, and the efficacy and the components of the rhizoma polygonati are not known. Therefore, the research is conducted on the brought-back rhizoma polygonati of different sources for comparison analysis, and the method for establishing the technical standard of the rhizoma polygonati of the traditional Chinese medicine is based on HPLC-HILIC-ELSD rhizoma polygonati polysaccharase hydrolysate analysis.

While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications or improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. The method for establishing the multi-element fingerprint of the polygonatum original species is characterized by comprising the following steps:

(2) Preparation of test solution: mixing a proper amount of rhizoma polygonati polysaccharide with fructosidase solution; hydrolyzing the mixture in an oscillator, heating, centrifuging, collecting supernatant, drying, and dissolving with acetonitrile/water solution;

gradient elution conditions: 0 to 25min,85 to 65 percent of A; 25-35 min, 65-50% of A.

2. The method of claim 1, wherein the control solution of step (1) is prepared by: taking 10mg of fructose, sucrose and glucose reference substances respectively, precisely weighing, transferring into a10 mL volumetric flask, and fixing the volume of water to a scale.

3. The method of claim 1, wherein the sample solution of step (2) is prepared by: adding water into rhizoma Polygonati polysaccharide to obtain rhizoma Polygonati polysaccharide solution with concentration of 0.5mg/mL, adding fructosidase solution with equal volume, mixing, placing into an oscillator for hydrolysis, heating, centrifuging, collecting supernatant, drying, and dissolving with 500 μl acetonitrile/water solution.

4. A method according to any one of claims 1 or 3, wherein the fructosidase solution in step (2) has a concentration of: 50-200U/mL.

5. The method of claim 4, wherein the fructosidase solution in step (2) has a concentration of: 100U/mL.

6. A method according to any one of claims 1 or 3, wherein the heating in step (2) is: heated at 80℃for 20 minutes.

7. A method according to any one of claims 1 or 3, wherein the setting conditions of the oscillator in step (2) are: the temperature is 55 ℃, the rotating speed is 200 revolutions per minute, and the time is 1-7 hours.

8. The method of claim 7, wherein the setting conditions of the oscillator in step (2) are: the temperature was 55℃and the rotational speed was 200 revolutions per minute for 3 hours.

9. A method according to any one of claims 1 or 3, wherein the centrifugation set-up conditions in step (2) are: 4500r/min,15min.

10. A method according to any one of claims 1 or 3, wherein the acetonitrile/water solution in step (2) is: the volume ratio is 1:1.