Quality detection method of radix isatidis polysaccharide

Abstract

The invention discloses a quality detection method of isatis root polysaccharide. The method comprises the following steps: identifying an ultraviolet spectrum and an infrared spectrum, establishing a hydrolyzed monosaccharide HPLC (High Performance Liquid Chromatography) fingerprint spectrum and an HPGPC (High Performance Liquid Chromatography Characteristic Chromatography) characteristic spectrum, and determining neutral polysaccharide, uronic acid, protein content and monosaccharide composition. The authenticity and the purity of the isatis root polysaccharide can be preliminarily judged through ultraviolet spectrum and infrared spectrum identification; the establishment of the hydrolyzed monosaccharide HPLC fingerprint spectrum and the HPGPC characteristic spectrum can be used for evaluating the consistency and the stability of the radix isatidis polysaccharide; the determination of neutral polysaccharide, uronic acid, protein content and monosaccharide composition can further accurately quantify the key components of the isatis root polysaccharide. The method disclosed by the invention is good in investigation result, has the advantages of high precision, good repeatability, strong stability and the like, can be effectively applied to quality detection of the isatis root polysaccharide, and provides powerful support for standardized production and quality control of the isatis root polysaccharide.

Description

Technical Field

[0001] The present invention relates to the quality detection technology of Isatis indigotica polysaccharide, specifically to a quality detection method and application of Isatis indigotica polysaccharide based on multi-dimensional spectra, belonging to the technical field of quality control and analysis of traditional Chinese medicine. Background Art

[0002] It has been found in research that free radicals play an important role in the process of causing senile degenerative diseases, and natural antioxidants may have important value in the prevention and treatment of senile complication diseases. For example, in diseases caused by various types of viruses, there are countless cases where the elderly die due to other complications caused by virus infections, and the drug treatments that can relieve the disease symptoms basically contain the ingredient of Isatis indigotica. Therefore, Isatis indigotica plays an important role in the treatment of diseases caused by various viruses.

[0003] As a traditional Chinese medicine, Isatis indigotica has the effects of clearing heat and detoxifying, cooling blood and relieving sore throat, etc., and is widely used in clinical practice. Isatis indigotica polysaccharide is one of its main active ingredients and has various pharmacological effects such as immune regulation, antiviral, and anti-inflammatory. With the continuous in-depth research on Isatis indigotica polysaccharide, its importance in the quality control of Isatis indigotica is becoming increasingly prominent.

[0004] At present, the quality control methods of Isatis indigotica mainly focus on the determination of the contents of ingredients such as indigo and indirubin, but these ingredients are not the effective ingredients for the main pharmacological effects of Isatis indigotica such as antiviral, and their contents in the water-soluble extract are very low. Therefore, using polysaccharide as the active index for the quality control of Isatis indigotica has gradually attracted attention. However, traditional methods have certain limitations in the quality detection of Isatis indigotica polysaccharide, such as being unable to comprehensively reflect the chemical characteristics and quality differences of polysaccharide.

[0005] Multi-dimensional spectrum technologies, such as high-performance liquid chromatography fingerprint and high-performance gel permeation chromatography fingerprint, etc., can provide more comprehensive and accurate chemical information, providing new ideas and methods for the quality detection of Isatis indigotica polysaccharide. Through multi-dimensional spectrum technologies, the monosaccharide composition, molecular weight distribution, etc. of Isatis indigotica polysaccharide can be analyzed in detail, so as to more accurately detect its quality. In addition, multi-dimensional spectrum technologies can also combine statistical methods such as cluster analysis and principal component analysis to analyze the quality differences of Isatis indigotica polysaccharide from different sources, providing a scientific basis for the quality control and standardized production of Isatis indigotica.

[0006] In summary, the research on the quality detection of Isatis indigotica polysaccharide based on multi-dimensional spectra can not only make up for the deficiencies of traditional methods, but also provide a more comprehensive and accurate detection means for the quality control of Isatis indigotica, having important research significance and application value. Summary of the Invention

[0007] Objective of the Invention: The present invention aims to provide a quality detection method for Isatis indigotica polysaccharide based on multi-dimensional spectrograms to achieve precise control of its quality.

[0008] Technical Solution: To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A quality detection method for Isatis indigotica polysaccharide, which comprises the following steps:

[0010] (1) Establishment of ultraviolet spectrum:

[0011] Take the Isatis indigotica polysaccharide sample, dissolve it in water to form a solution, use pure water as the blank, and perform a full-wavelength scan from 200 to 800 nm with an ultraviolet scanner to draw an ultraviolet spectrum;

[0012] (2) Establishment of infrared spectrum:

[0013] Take the Isatis indigotica polysaccharide sample, mix it with KBr in proportion, grind and make a tablet, and scan it with a Fourier transform infrared spectrometer. The spectral range is mid-infrared 4000 - 400 cm -1 , and draw an infrared spectrum;

[0014] (3) Establishment of HPLC fingerprint of hydrolyzed monosaccharides:

[0015] Weigh the Isatis indigotica polysaccharide sample into an ampoule bottle, add trifluoroacetic acid, heat and hydrolyze it to obtain hydrolyzed Isatis indigotica polysaccharide;

[0016] Take the hydrolyzed Isatis indigotica polysaccharide, monosaccharide mixed solution, and monosaccharide solution, add NaOH solution, add PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution, mix well, heat in a water bath, after cooling, add HCl solution to neutralize the pH value, shake well, add chloroform for extraction, take the supernatant, filter it through a 0.22 μm microporous filter membrane, and reserve it. Perform HPLC analysis according to the set chromatographic conditions to establish a fingerprint;

[0017] (4) Establishment of HPGPC characteristic spectrum: Weigh the Isatis indigotica polysaccharide sample into a volumetric flask, dissolve it with sodium nitrate to prepare a polysaccharide solution, filter it through a 0.22 μm filter membrane, and collect the HPGPC spectrum according to the set high-performance gel permeation chromatography method to generate a fingerprint;

[0018] (5) Determination of the contents of neutral polysaccharide, uronic acid, and protein: Weigh the Isatis indigotica polysaccharide sample precisely, and respectively use the phenol-sulfuric acid method, m-hydroxybiphenyl method, and Coomassie brilliant blue method to determine the contents of neutral polysaccharide, uronic acid, and protein;

[0019] (6) Determination of monosaccharide composition: Take the acid-hydrolyzed Isatis indigotica polysaccharide sample, perform PMP derivatization-HPLC analysis according to the set chromatographic conditions to determine the monosaccharide composition.

[0020] The quality detection method of the above-mentioned isatis root polysaccharide, step (1) establishment of ultraviolet spectrum:

[0021] Take samples of isatis root polysaccharide from different batches, dissolve them in water to form a solution, use pure water as the blank, perform a full-wavelength scan from 200 to 800 nm with an ultraviolet scanner, and use Origin 2022 software to draw the ultraviolet spectra of 13 batches of isatis root polysaccharide;

[0022] Step (2) establishment of infrared spectrum:

[0023] Take samples of isatis root polysaccharide from different batches and grind them with KBr in a ratio of 1:100 to make tablets, scan with a Fourier transform infrared spectrometer, and the spectral range is mid-infrared 4000 - 400 cm -1 , and use Origin 2022 software to draw the infrared spectra of 13 batches of isatis root polysaccharide.

[0024] Step (3) establishment of HPLC fingerprint of hydrolyzed monosaccharides:

[0025] Take the isatis root polysaccharide sample into an ampoule, add trifluoroacetic acid, seal it with an alcohol burner, perform acid hydrolysis in an oven at 110 - 120 °C for 4 - 6 h, add methanol and repeatedly volatilize until trifluoroacetic acid is completely volatilized, add ultrapure water to redissolve, take the hydrolyzed isatis root polysaccharide, monosaccharide mixed solution, and monosaccharide solution, add NaOH solution, add PMP methanol solution, mix well, heat in a water bath, cool, add HCl solution to neutralize the pH, shake well, extract with chloroform, take the supernatant, filter it through a 0.22 μm microporous filter membrane for standby, perform HPLC analysis according to the set chromatographic conditions to obtain a chromatogram, import it into the "Similarity Detection System for Traditional Chinese Medicine Chromatographic Fingerprints" software version 2012 in CDF format, synthesize the fingerprint, use S1 as the reference peak map, generate a control map by the "average method", set the time width to 0.1 min, perform multi-point calibration and Mark peak matching on the isatis root polysaccharide chromatographic peaks, and establish the fingerprint.

[0026] The isatis root polysaccharide sample is prepared by the following method: Take 2 kg of isatis root decoction pieces, add 8 times the amount of water and reflux extract twice, each time for 3 h, combine the two water decoctions, and concentrate under reduced pressure until each 1 mL of concentrated solution contains 1 g of crude drug content, add 95% ethanol, stir rapidly to make the final alcohol concentration in the system 80%, stand for alcohol precipitation overnight in a cool place, vacuum filter to remove the supernatant, and vacuum dry to obtain crude polysaccharide. Take 20 g of crude polysaccharide (IRPS) and redissolve it in water, and perform enzymatic deproteinization according to an enzyme activity of 500 U·mL -1 , pH = 8.0, enzymatic hydrolysis time of 5 h, and enzymatic hydrolysis temperature of 37 °C. After the reaction is completed, centrifuge. What is obtained in this step is an aqueous solution of deproteinized polysaccharide, and then it is repeatedly freeze-thawed and concentrated and vacuum dried to obtain isatis root polysaccharide.

[0027] As a preferred solution: for the quality detection method of the isatis root polysaccharide described above, the steps for determining the similarity by HPGPC method in step (4) include: taking an isatis root polysaccharide sample, dissolving it with sodium nitrate to prepare an isatis root polysaccharide solution with a certain concentration, centrifuging and filtering through a microporous membrane, then collecting the HPGPC spectrum of the isatis root polysaccharide by high performance gel permeation chromatography, importing it into the "Similarity Detection System for Traditional Chinese Medicine Chromatographic Fingerprints" software version 2012 in CDF format, synthesizing the fingerprint spectrum, and calculating the similarity of the isatis root polysaccharide.

[0028] Among them, the chromatographic conditions for high performance gel permeation chromatography are as follows: Waters 2414 differential refractive index detector (refractive index detector, RID); the chromatographic column is a gel column TSK-gel G5000PW in series with TSK-gel G3000PW (8.0×300mm, 6μM); the mobile phase: 0.1mol·L -1 Sodium nitrate; the flow rate is 0.4mL·min -1 ; the column temperature is 35°C; the injection volume is 10μL.

[0029] The method for determining the content of neutral polysaccharide in step (5) includes the following steps

[0030] (1) Drawing of glucose standard curve

[0031] Precisely weigh D-anhydrous glucose, make up the volume, precisely measure 0, 0.05, 0.1, 0.15, 0.2, 0.3mL respectively, place them in stoppered test tubes, add pure water to make up to 1.0mL, add 0.5mL of 5% phenol solution, 2.5mL of concentrated sulfuric acid and shake well, heat in a water bath at 100°C for 20 minutes, after cooling, measure the absorbance A at a wavelength of 490nm as the ordinate and the glucose concentration C as the abscissa to draw the standard curve;

[0032] (2) Sample determination

[0033] Precisely weigh the isatis root polysaccharide in a volumetric flask, prepare a test solution of the isatis root polysaccharide with pure water, measure its absorbance value according to the method described in step (1), and substitute it into the standard curve to calculate the content;

[0034] The determination of uronic acid content includes the following steps

[0035] (1) Solution preparation

[0036] Sodium tetraborate - concentrated sulfuric acid solution: weigh sodium tetraborate, add concentrated sulfuric acid, and dissolve it fully;

[0037] 0.15% m-hydroxybiphenyl solution: weigh m-hydroxybiphenyl in a volumetric flask, dissolve it with 5% sodium hydroxide solution and make up to the scale;

[0038] (2) Preparation of Glucuronic Acid Standard Curve

[0039] Precisely weigh D - glucuronic acid, make up the volume to obtain the D - glucuronic acid standard solution. Precisely measure 0, 0.1, 0.2, 0.4, 0.6, 0.7 mL of the standard solution respectively and place them in stoppered test tubes. Add pure water to make up to 1.0 mL. Add 6 mL of sodium tetraborate - concentrated sulfuric acid and 0.1 mL of m - hydroxybiphenyl solution, shake well, let stand at room temperature for 40 minutes, and plot the standard curve with the absorbance value A measured at 525 nm as the ordinate and the glucuronic acid concentration C as the abscissa;

[0040] (3) Determination of Samples

[0041] Precisely weigh the polysaccharide from Isatis indigotica Fort. in a volumetric flask, prepare the test solution of the polysaccharide from Isatis indigotica Fort. with pure water, make up with pure water, and measure its absorbance value according to the method in step (2) above, and calculate the content by substituting into the standard curve;

[0042] Determination of Protein Content

[0043] (1) Preparation of Solutions

[0044] Test sample solution: Take the polysaccharide from Isatis indigotica Fort. in a volumetric flask, dissolve it with pure water and make up to the mark to obtain the test sample solution of the polysaccharide from Isatis indigotica Fort.;

[0045] Coomassie Brilliant Blue G250 solution: Precisely weigh Coomassie Brilliant Blue G250 powder in a volumetric flask, add ethanol, add H3PO4 solution, and make up the volume;

[0046] (2) Preparation of Protein Standard Curve

[0047] Precisely weigh bovine serum albumin, dissolve it with water and make up the volume. Precisely measure 0, 0.1, 0.2, 0.5, 0.6, 0.7 mL of the standard solution respectively and place them in stoppered test tubes. Add pure water to make up to 1.0 mL. Add 5 mL of Coomassie Brilliant Blue G250 solution, shake well, let stand at room temperature in the dark for 5 minutes, and plot the standard curve with the absorbance value A measured at 595 nm by an ELISA reader as the ordinate and the bovine serum albumin concentration C as the abscissa;

[0048] (3) Determination of Samples

[0049] Take 1 mL of the test sample solution of the polysaccharide from Isatis indigotica Fort. obtained in step (1), add 5 mL of Coomassie Brilliant Blue G250 solution, shake well, let stand at room temperature for 5 minutes, measure the absorbance at 595 nm by an ELISA reader, and calculate the content by substituting into the standard curve in step (2).

[0050] Step (6) The determination of monosaccharide content includes the following steps:

[0051] Precisely weigh the polysaccharide sample from Isatis indigotica Fort. into an ampoule, add 2 mol·L -1Trifluoroacetic acid was used, sealed with an alcohol blowtorch, reacted in an oven at 110 °C for 4 h, the trifluoroacetic acid was removed by rotary evaporation, redissolved with methanol, rotary evaporated until the trifluoroacetic acid was completely evaporated, and redissolved with ultrapure water to obtain the Isatis indigotica polysaccharide sample solution;

[0052] Mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, arabinose, and galactose were accurately weighed and prepared into a mixed reference solution with a certain concentration with ultrapure water for standby;

[0053] Take 500 μL of the mixed reference solution and the Isatis indigotica polysaccharide sample solution respectively, add 75 μL of 0.3 mol·L -1 NaOH solution, then add 150 μL of 10% PMP methanol solution, vortex and mix well, heat in a water bath at 70 °C for 1 h, cool to room temperature, add 75 μL of 0.3 mol·L -1 HCl solution to neutralize the pH, shake and extract with chloroform 3 times, take the supernatant, filter through a 0.22 μm microporous membrane for analysis;

[0054] (2) Investigation of linear relationship

[0055] Take the mixed reference solution after PMP derivatization in step (1) as the first concentration, and gradually dilute it into a series of standard solutions. Inject continuously without interruption. Take the peak area as the ordinate and the concentration as the abscissa, plot the standard curve and perform linear regression;

[0056] The chromatographic conditions are as follows: AlltimaTM C18 chromatographic column (250 mm × 4.6 mm, 5 μm); the mobile phase is 0.1 mol·L -1 Phosphate buffer solution (pH 6.8) - acetonitrile (83:17), isocratic elution; column temperature 30 °C; volume flow rate 1.0 mL·min -1 ; injection volume 10 μL, detection wavelength 245 nm. Inject continuously without interruption. Take the peak area as the ordinate and the concentration as the abscissa, plot the standard curve and perform linear regression;

[0057] (3) Determination of monosaccharide content

[0058] Take the Isatis indigotica polysaccharide sample in step (1) and inject it according to the chromatographic conditions in step (2), and substitute it into the standard curve in step (2) to calculate the monosaccharide content in the Isatis indigotica polysaccharide.

[0059] The present invention has the following advantages:

[0060] The present invention comprehensively uses ultraviolet spectroscopy, infrared spectroscopy identification, establishment of hydrolyzed monosaccharide HPLC fingerprint, HPGPC characteristic fingerprint, and determination of neutral polysaccharide, uronic acid, protein content and monosaccharide composition, etc. to analyze the isatis root polysaccharide from different dimensions, which can comprehensively and accurately reflect its quality characteristics and provide rich and reliable basis for quality control. The experimental results show that the content of neutral polysaccharide in the isatis root polysaccharide is between 55.36% and 79.38%, the content of uronic acid is between 14.74% and 20.82%, the protein content is less than 2.74%, and it is mainly composed of 5 neutral monosaccharides and 1 uronic acid.

[0061] The present invention can provide strong support for the standardized production of isatis root polysaccharide, help enterprises effectively monitor and control the product quality in the production process, improve the stability and consistency of product quality, and promote the standardized development of the isatis root polysaccharide industry. Description of the Drawings

[0062] Figure 1 They are the ultraviolet and infrared spectra of 13 batches of isatis root polysaccharide.

[0063] Figure 2 They are the hydrolyzed monosaccharide HPLC fingerprints (254nm) of 13 batches of isatis root polysaccharide.

[0064] Figure 3 They are the HPGPC fingerprints of 13 batches of isatis root polysaccharide.

[0065] Figure 4 They are the HPGPC characteristic fingerprints of 13 batches of isatis root polysaccharide.

[0066] Figure 5 They are the separation effects of 7 monosaccharides. Detailed Embodiments

[0067] The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art, and the raw materials used are all commercially available products or raw materials.

[0068] Example 1 Preparation of Isatis Root Polysaccharide

[0069] Take 200 g of 13 batches of isatis root decoction pieces, add 8 times of water and reflux for extraction twice, 3 h each time. Combine the two water decoctions and concentrate under reduced pressure to 1 g of crude drug per 1 mL of concentrated solution. Add 95% ethanol, stir slowly and then quickly to make the final alcohol concentration in the system 80%. Let it stand overnight for alcohol precipitation in a cool place, vacuum filter to remove the supernatant, and vacuum dry to obtain the crude polysaccharide. Take 20 g of the crude polysaccharide and redissolve it in water, with an enzyme activity of 500 U·mL -1, pH = 8.0, the enzymatic hydrolysis time was 5 h, and the enzymatic hydrolysis temperature was 37 °C for enzymatic deproteinization. After the reaction was completed, centrifugation was carried out. The aqueous solution of deproteinized polysaccharide was obtained in this step, and then freeze-thawed repeatedly. After vacuum drying, 13 batches of Isatis indigotica polysaccharide were collected for standby. The Isatis indigotica medicinal materials were purchased from different markets across the country and were identified as the dried roots of Isatis tinctoria, a cruciferous plant, by Professor Duan Jin'ao of Nanjing University of Chinese Medicine. The detailed information and numbers of the medicinal materials are shown in Table 1.

[0070] Table 1 Information Table of Medicinal Materials

[0071] Number Place of origin Company Batch number S1 Anhui Jiangxi Pengshi Guoyao Hall Decoction Pieces Co., Ltd. 21122712 S2 Hebei Jiangxi Pengshi Guoyao Hall Decoction Pieces Co., Ltd. 22042014 S3 Shanxi Jiangxi Guodu Chinese Medicine Decoction Pieces Co., Ltd. 20201101 S4 Jiangsu Suzhou Tianling Chinese Medicine Decoction Pieces Co., Ltd. 210601 S5 Nanjing, Jiangsu Pharmacy Garden of Nanjing University of Chinese Medicine 20211116 S6 Inner Mongolia Xing'an League Bozhou Zirui Pharmaceutical Co., Ltd. 248211218 S7 Hami, Xinjiang Bozhou Zirui Pharmaceutical Co., Ltd. 248211116 S8 Daqing, Heilongjiang Bozhou Zirui Pharmaceutical Co., Ltd. 248210122 S9 Lindian, Heilongjiang Bozhou Zirui Pharmaceutical Co., Ltd. 248210123 S10 Jiuquan, Gansu Bozhou Zirui Pharmaceutical Co., Ltd. 258210808 S11 Minle, Gansu Bozhou Zirui Pharmaceutical Co., Ltd. 248210230 S12 Wuwei, Gansu Bozhou Zirui Pharmaceutical Co., Ltd. 248210411 S13 Gaotai, Gansu Bozhou Zirui Pharmaceutical Co., Ltd. 248210453

[0072] Example 2 Establishment of Ultraviolet Spectrum

[0073] Take the 13 batches of Isatis indigotica polysaccharide samples from Example 1 and dissolve them in water to prepare a solution of 1 mg·mL -1 . Using pure water as the blank, perform a full-wavelength scan from 200 to 800 nm with an ultraviolet scanner, and use Origin 2022 software to draw the ultraviolet spectra of 13 batches of Isatis indigotica polysaccharide.

[0074] The scanning results of 13 batches of Isatis indigotica polysaccharide are as Figure 1 (a). There are no absorption peaks at 200 - 300 nm for 13 batches of Isatis indigotica polysaccharide, indicating that 13 batches of Isatis indigotica polysaccharide do not contain impurities such as proteins, nucleic acids, and amino acids.

[0075] Example 3 Establishment of Infrared Spectrum

[0076] Take 13 batches of Isatis indigotica polysaccharide samples and grind them with KBr in a ratio of 1:100 to make tablets, and scan them with a Fourier transform infrared spectrometer. The spectral range is mid-infrared 4000 - 400 cm -1 . Use Origin 2022 software to draw the infrared spectra of 13 batches of Isatis indigotica polysaccharide.

[0077] The infrared scanning results of 13 batches of Isatis indigotica polysaccharide are as Figure 1 (b). The peak shapes and characteristic peaks of the infrared spectra of 13 batches of Isatis indigotica polysaccharide are consistent. The peak near 3424 cm -1 is the characteristic peak of polysaccharide hydroxyl groups, caused by the stretching vibration of O - H. The peak around 2950 cm -1 is caused by the vibration of C - H of methyl and methylene groups. The peak at 1622 cm -1 is caused by the CO conjugation of the carbonyl group. The absorption peak near 1400 cm -1 is caused by the bending vibration of C - H. The peak at 1064 cm -1 is attributed to the stretching vibration absorption peak of the C-(C)-O bond of polysaccharide. The characteristic peaks belonging to furanose are still present in the range of 900 - 700 cm -1 . The results show that 13 batches of Isatis indigotica polysaccharide conform to the infrared characteristics of polysaccharide.

[0078] Example 4 Establishment of HPLC Fingerprint of Hydrolyzed Monosaccharides

[0079] 1. Sample Preparation

[0080] Weigh 20 mg of 13 batches of Isatis indigotica polysaccharide samples into an ampoule, add 2 mL of 2 mol·L -1 TFA, seal it with an alcohol burner, acid hydrolyze it in an oven at 110 °C for 4 h, add 1 mL of methanol and evaporate repeatedly until the TFA is completely evaporated, then add 2 mL of ultrapure water to redissolve. Divide it into 2 parts, which are used for the establishment of HPLC fingerprint and content determination respectively. Take 0.5 mL of the hydrolyzed 13 batches of Isatis indigotica polysaccharide, monosaccharide mixed solution and monosaccharide solution respectively, add 75 μL of 0.3 mol·L -1 NaOH solution, add 150 μL of 10% PMP methanol solution, mix well, heat in a water bath at 70 °C for 1 h, after cooling, add 75 μL of 0.3 mol·L -1 HCl solution to neutralize the pH, shake well and extract with chloroform 3 times, take the supernatant, filter it through a 0.22 μm microporous filter membrane for standby.

[0081] 2. Chromatographic Conditions

[0082] AlltimaTM C18 chromatographic column (250 mm × 4.6 mm, 5 μm); the mobile phase is 0.1 mol·L -1 phosphate buffer solution (pH 6.8 - acetonitrile (83∶17), isocratic elution; column temperature 30 °C; volume flow rate 1.0 mL·min -1 ; injection volume 10 μL, detection wavelength 245 nm.

[0083] 3. Methodology Investigation

[0084] Precision Test: Inject the hydrolyzed sample solution of S1 Isatis indigotica polysaccharide continuously for 6 times according to the above chromatographic conditions to collect chromatogram data. Using the glucose peak at No. 4 as the reference peak, calculate the RSD values of the relative retention time and relative peak area of other common peaks respectively. The results are shown in Table 2. The RSD values of the relative retention time of 6 common peaks of Isatis indigotica polysaccharide are all less than 0.91%, and the RSD values of the relative peak area are all less than 2.46%, indicating that the precision of the instrument used is good.

[0085] Repeatability Test: Take 6 portions of the hydrolyzed sample of S1 Isatis indigotica polysaccharide, collect chromatogram data according to the above chromatographic conditions. Using the glucose peak at No. 4 as the reference peak, calculate the relative retention time and peak area of other common peaks respectively. The results are shown in Table 2. The RSD values of the relative retention time of each common peak are all less than 0.78%, and the RSD values of the relative peak area are all less than 2.86%, indicating that the method adopted in this experiment has good repeatability.

[0086] Stability test: Take the prepared hydrolyzed test solution of S1 polysaccharide from Isatis indigotica Fort., inject samples at 0, 2, 4, 8, 12, and 24 h respectively, collect chromatogram data according to the above chromatographic conditions. Using glucose at peak 4 as the reference peak, calculate the relative retention time and peak area of other common peaks. The results are shown in Table 2. The RSD value of the relative retention time of each common peak is less than 0.71%, and the RSD of the relative peak area is less than 4.62%, indicating that after the polysaccharide from Isatis indigotica Fort. is derivatized with PMP, the test solution has good stability within 24 h.

[0087] Table 2 Results of methodology investigation

[0088]

[0089] 4. Establishment of fingerprint and generation of reference chromatogram

[0090] Derivatize the acid hydrolysis solutions of the above 13 batches of polysaccharide from Isatis indigotica Fort., collect chromatograms according to the above chromatographic conditions, and import them into the software "Similarity Detection System for Traditional Chinese Medicine Chromatographic Fingerprints" (2012 version) in CDF format to synthesize fingerprint chromatograms. Using the chromatogram of S1 as the reference peak chromatogram, generate a reference chromatogram by the "average method", set the time width to 0.1 min, perform multi-point calibration and Mark peak matching on the chromatographic peaks of 13 batches of polysaccharide from Isatis indigotica Fort. to generate fingerprint chromatograms, and calculate the similarity.

[0091] 5. Results

[0092] 5.1 Establishment of HPLC fingerprint

[0093] The HPLC fingerprints of hydrolyzed monosaccharides of 13 batches of polysaccharide from Isatis indigotica Fort. are as Figure 2 . The fingerprint chromatograms generated a total of 6 common peaks. After identification with reference standards, peaks 1 - 6 are mannose, rhamnose, galacturonic acid, glucose, arabinose, and galactose in sequence. Since the glucose at chromatographic peak 4 has the largest peak area in all samples, peak 4 is used as the reference peak (S) to calculate the relative retention time and relative peak area of each common peak. The results are shown in Tables 3 and 4. The results show that the RSD% values of the relative retention time of each chromatographic peak are all less than 0.44%, and the RSD% values of the relative peak area are from 32.37% to 90.46%, with relatively large differences, indicating that the elution times of the 6 common monosaccharides in 13 batches of polysaccharide from Isatis indigotica Fort. are relatively stable, but the contents vary greatly. The content difference of galacturonic acid is the largest, followed by arabinose and galactose, while the contents of rhamnose and mannose are relatively small, with relatively small differences.

[0094] Table 3 Relative retention time of 6 common peaks

[0095]

[0096] Table 4 Relative peak area of 6 common peaks

[0097]

[0098] 5.2 HPLC Fingerprint Similarity Detection

[0099] The results of similarity calculation and comparison between the HPLC fingerprints of the hydrolyzed monosaccharides of polysaccharides from 13 batches of Isatis indigotica Fort. and the control fingerprint showed that S1 was the control fingerprint, and the similarities of S1 - S13 were 1.000, 0.999, 0.999, 0.998, 0.999, 0.999, 0.994, 1.000, 0.999, 1.000, 0.999, 0.971, 0.999 respectively, indicating relatively good consistency among batches and stable and reliable fingerprints. It can provide a theoretical reference for the identification of the authenticity, quality, and inferiority of Isatis indigotica Fort. in the market.

[0100] Example 5 Establishment of HPGPC Characteristic Fingerprint

[0101] Weigh 2 mg of the polysaccharide samples from 13 batches of Isatis indigotica Fort. into a 2 - mL volumetric flask, and dissolve them with 0.1 mol·L -1 sodium nitrate to prepare a 2 mg·mL -1 polysaccharide solution. Filter it through a 0.22 - μm filter membrane. According to the high - performance gel permeation chromatography method: the detector is a Waters 2414 refractive index detector (RID); the chromatographic column is a gel column composed of TSK - gel G5000PW in series with TSK - gel G3000PW (8.0×300 mm, 6 μM); the mobile phase is 0.1 mol·L -1 sodium nitrate; the flow rate is 0.4 mL·min -1 ; the column temperature is 35°C; the injection volume is 10 μL.

[0102] Collect the HPGPC fingerprints of the polysaccharides from 13 batches of Isatis indigotica Fort., and import them into the "Similarity Detection System for Traditional Chinese Medicine Chromatographic Fingerprints" software (2012 version) in CDF format to synthesize the fingerprint. Using S1 as the reference peak fingerprint, generate the control fingerprint by the "average method", set the time width to 0.1 min, perform multi - point calibration and Mark peak matching on the chromatographic peaks of 13 batches of Isatis indigotica Fort. polysaccharides to generate the fingerprint, and calculate the similarity.

[0103] 1. Establishment of HPGPC Characteristic Fingerprint

[0104] The HPGPC characteristic fingerprints of the polysaccharides from 13 batches of Isatis indigotica Fort. are as Figure 4 shown, and the results of the control fingerprint are shown in Figure 3(S1 to S13: 13 batches of samples; R: reference chromatogram). A total of 6 common peaks were generated in the characteristic chromatogram. According to the retention time, they were sorted in order. Taking the 3rd peak with the highest peak area as the reference peak, the relative retention time and relative peak area results of each peak are shown in Tables 5 and 6. The results showed that the RSD% value of the relative retention time of each chromatographic peak was less than 1.68%, and the RSD% values of the relative peak areas were 60.5885%, 27.4646%, 0.0000%, 24.2329%, 25.0766%, and 44.5087% in sequence, indicating that the molecular weight sizes of the components contained in the polysaccharides of 13 batches of Isatis indigotica Fort. were not very different, but there were differences in the component distributions of each molecular weight.

[0105] 2. Similarity detection of HPGPC characteristic chromatogram

[0106] Referring to the reference chromatogram, the similarities of the HPGPC characteristic chromatograms S1 to S13 of 13 batches of Isatis indigotica Fort. were 0.998, 0.928, 0.998, 0.993, 0.988, 0.999, 0.998, 0.991, 0.992, 0.998, 0.999, 0.987, and 0.998 in sequence, indicating that the consistency among batches was relatively good, the fingerprint chromatogram was stable and reliable, and it could provide a theoretical reference for the identification of the authenticity, quality, and inferiority of Isatis indigotica Fort. in the market.

[0107] Table 5 Relative retention time of 6 common peaks

[0108]

[0109] Table 6 Relative peak area of 6 common peaks

[0110]

[0111] Example 6 Determination of the content of polysaccharides in Isatis indigotica Fort.

[0112] 1. Materials and instruments

[0113] 1.1 Instruments

[0114] UV-2600 ultraviolet spectrophotometer (Shimadzu Corporation, Japan); FT-IR Fourier transform infrared spectrometer (PerkinElmer Co., Ltd., USA); high performance liquid chromatograph (Waters Corporation, USA); BUCHI B-300 rotary evaporator (Yuhua Instrument Co., Ltd., Gongyi City); D2012 high-speed tabletop centrifuge (Dalong Xingchuang Experimental Instrument Co., Ltd., Beijing); HH-6 digital display constant temperature water bath (Guohua Instrument Manufacturing Co., Ltd.); X025-12DT ultrasonic cleaner (Nanjing Xianou Instrument Co., Ltd.).

[0115] 1.2 Reagents

[0116] Bovine serum albumin standard (Beijing Solarbio Science & Technology Co., Ltd., 128K053); Coomassie Brilliant Blue G-250 (Biosharp, 21260625), trypsin, papain, bromelain (Shanghai Yuanye Bio-Technology Co., Ltd., L22A8Y34505, M05GS140428, P09N11B130551); Pepsin (Sinopharm Chemical Reagent Co., Ltd., 20211103); Glucose, DL-Arabinose, D-Mannose (Chengdu Lemeitian Medical Science Co., Ltd., DSTDW000501, DSTDA003801, DSTDG004701); D-Galacturonic acid, D-Glucuronic acid, Rhamnose (K07J12B133073, 27GB155998, O27GS165538); 1-Phenyl-3-methyl-5-pyrazolone (Solarbio, 1029D022); Trifluoroacetic acid (Shanghai Macklin Biochemical Co., Ltd., C10672531); Acetonitrile (chromatographic grade, Anhui Tiandi Co., Ltd.).

[0117] 1.3 Medicinal Materials

[0118] Same as "Example 1".

[0119] 2 Methods

[0120] 2.1 Preparation of Test Sample Solution

[0121] Precisely weigh 10 mg of 13 batches of Isatis indigotica polysaccharide prepared by the method of Example 1 into a 10 mL volumetric flask, and prepare a test solution with a concentration of 1 mg / mL using pure water.

[0122] 2.2 Determination of Neutral Polysaccharide Content

[0123] 2.2.1 Preparation of Glucose Standard Curve

[0124] Precisely weigh 15.61 mg of D-anhydroglucose and make up the volume to 10 mL to obtain a standard solution containing 1.561 mg of anhydrous glucose per 1 mL. Precisely measure 0, 0.05, 0.1, 0.15, 0.2, 0.3 mL, respectively, place them in 15 mL stoppered test tubes, add pure water to make up to 1.0 mL, add 0.5 mL of 5% phenol solution, 2.5 mL of concentrated sulfuric acid and shake well. Heat in a water bath at 100 °C for 20 minutes. After cooling, measure the absorbance (A) at a wavelength of 490 nm as the ordinate and the glucose concentration (C) as the abscissa to draw a standard curve.

[0125] 2.2.2 Sample Determination

[0126] Take 0.5 mL of the test solution of Radix Isatidis polysaccharides from batch 13, add pure water to make up to 1 mL, determine its absorbance value according to the method described in "2.2.1", and substitute it into the standard curve to calculate the content.

[0127] 2.2.3 Methodology

[0128] Precision: Accurately pipette 6 portions of glucose solution, determine the absorbance value according to the method described in "2.2.1", and calculate the content RSD%.

[0129] Repeatability: Accurately weigh 6 portions of S10 polysaccharide sample, determine the absorbance value according to the method described in "2.2.1", and calculate the content RSD%.

[0130] Stability: Take 1 portion of S10 polysaccharide and complete the reaction according to the method described in "2.2.1". Then measure the absorbance at 0, 15, 30, 60, 90 and 120 minutes and calculate the RSD% value of the content.

[0131] Sample recovery: Take 6 portions of S10 polysaccharide, add glucose standard with the same content, determine the absorbance value according to the method described in "2.2.1", and calculate the RSD% of the sample recovery rate.

[0132] 2.3 Determination of uronic acid content

[0133] 2.3.1 Solution preparation

[0134] Sodium tetraborate-concentrated sulfuric acid solution: Weigh 1.44 g of sodium tetraborate, add 300 mL of concentrated sulfuric acid and fully dissolve.

[0135] 0.15% m-hydroxybiphenyl solution: weigh 75 mg of m-hydroxybiphenyl into a 50 mL volumetric flask, add 5% sodium hydroxide solution to dissolve and dilute to the mark.

[0136] 2.3.2 Drawing of glucuronic acid standard curve

[0137] Weigh 10.62 mg of D-glucuronic acid accurately and make it to 10 mL, so that each 1 mL of standard solution contains 1.062 mg of glucuronic acid. Accurately measure 0, 0.1, 0.2, 0.4, 0.6, and 0.7 mL of standard solution, respectively, and place them in 15 mL stoppered test tubes, add pure water to make up to 1.0 mL, add 6 mL of sodium tetraborate-concentrated sulfuric acid and 0.1 mL of m-hydroxybiphenyl solution, shake well, and place at room temperature for 40 minutes. Measure the absorbance value (A) at 525 nm as the ordinate and the glucuronic acid concentration (C) as the abscissa to draw a standard curve.

[0138] 2.3.3 Sample determination

[0139] Take 0.25 mL of the test solution of Radix Isatidis polysaccharides from batch 13, add pure water to make up to 1 mL, determine its absorbance value according to the "2.3.2" method, and substitute it into the standard curve to calculate the content.

[0140] 2.3.4 Methodology

[0141] Precision: Accurately pipette 6 portions of glucuronic acid solution, determine the absorbance value according to the method described in "2.3.2", and calculate the content RSD%.

[0142] Repeatability: Accurately weigh 6 portions of the same polysaccharide sample, determine the absorbance value according to the method described in "2.3.2", and calculate the RSD% of the content.

[0143] Stability: Take one portion of the test sample and complete the reaction according to the method described in "2.3.2". Then measure the absorbance at 0, 15, 30, 60, 90 and 120 minutes and calculate the RSD% value of the content.

[0144] Sample recovery: Take 6 portions of the same polysaccharide, add glucuronic acid standard with the same content, determine the absorbance value according to the method described in "2.3.2", and calculate the RSD% of the sample recovery rate.

[0145] 2.4 Protein content determination

[0146] 2.4.1 Solution preparation

[0147] Test sample solution: Take 5 mg of Radix Isatidis polysaccharide from batch 13 into a 10 mL volumetric flask, add pure water to dissolve and then dilute to the mark.

[0148] Coomassie Brilliant Blue G250 solution: Accurately weigh 5 mg of Coomassie Brilliant Blue G250 powder into a 50 mL volumetric flask, add 2.5 mL of 95% ethanol, add 5 mL of 85% H3PO4 solution, and dilute to 50 mL.

[0149] 2.4.2 Plotting of protein standard curve

[0150] Accurately weigh 5.01 mg of bovine serum albumin, dissolve it in water to make up to 25 mL, accurately measure 0, 0.1, 0.2, 0.5, 0.6, and 0.7 mL of standard solution, respectively place them in 15 mL stoppered test tubes, add pure water to make up to 1.0 mL, add 5 mL of Coomassie Brilliant Blue G250 solution, shake well, and place it at room temperature for 5 minutes away from light. Measure the absorbance value (A) at a wavelength of 595 nm on a microplate reader as the ordinate, and draw a standard curve with the bovine serum albumin concentration (C) as the abscissa.

[0151] 2.4.3 Sample determination

[0152] Take 1 mL of the test solution of Radix Isatidis polysaccharides from batch 13, add 5 mL of Coomassie Brilliant Blue G250 solution, shake well, leave at room temperature for 5 minutes, measure the absorbance at a wavelength of 595 nm on an enzyme reader, and substitute it into the standard curve to calculate the content.

[0153] 2.4.4 Methodology

[0154] Precision: Accurately pipette 6 portions of bovine serum albumin solution, determine the absorbance value according to the method described in "2.4.3", and calculate the content RSD%.

[0155] Repeatability: Accurately weigh 6 portions of the same polysaccharide sample, determine the absorbance value according to the method described in "2.4.3", and calculate the RSD% of the content.

[0156] Stability: Take one portion of the test sample and complete the reaction according to the method described in "2.4.3". Then measure the absorbance at 0, 15, 30, 60, 90 and 120 minutes and calculate the RSD% value of the content.

[0157] Sample recovery: Take 6 portions of the same polysaccharide, add bovine serum albumin standard with the same content, determine the absorbance value according to the method described in "2.4.3", and calculate the RSD% of the sample recovery rate.

[0158] 2.5 Determination of monosaccharide content

[0159] 2.5.1 Sample preparation

[0160] Accurately weigh 10 mg of Radix Isatidis polysaccharide sample into an ampoule and add 2 mol·L -1 Trifluoroacetic acid (TFA) was melted and sealed with an alcohol blowtorch, and the mixture was reacted in an oven at 110°C for 4 h. TFA was removed by rotary evaporation, and 1 mL of methanol was added for re-dissolution. The mixture was rotary evaporated until TFA was evaporated to dryness, and 1 mL of ultrapure water was added for re-dissolution to obtain a Radix Isatidis polysaccharide sample solution.

[0161] Accurately weigh 1.48 mg mannose, 1.28 mg rhamnose, 1.18 mg glucuronic acid, and 1.6 mg galacturonic acid.

[0162] 1.59 mg, 2.9 mg of glucose, 1.91 mg of arabinose, and 1.66 mg of galactose are placed in a 1 mL volumetric flask, and water is added to the mark to make the monosaccharide standard solution. Take 0.1 mL of each and mix them to make the mixed reference solution.

[0163] Take 500 μL of the mixed reference solution and Radix Isatidis polysaccharide sample solution, add 0.3 mol·L -1 75 μL of NaOH solution was added to 150 μL of 10% PMP methanol solution, vortexed and mixed, placed in a 70°C water bath for 1 h, cooled to room temperature, and added 0.3 mol·L -1Neutralize the pH with 75 μL of HCl solution, add chloroform and extract 3 times after shaking. Take the supernatant and filter it through a 0.22 μm microporous membrane to obtain the test sample.

[0164] 2.5.2 Chromatographic conditions

[0165] AlltimaTM C18 chromatographic column (250 mm × 4.6 mm, 5 μm); the mobile phase is 0.1 mol·L -1 Phosphate buffer (pH 6.8) - acetonitrile (83:17), isocratic elution; column temperature 30 °C; flow rate 1.0 mL·min -1 ; injection volume 10 μL, detection wavelength 245 nm.

[0166] 2.5.3 Investigation of linear relationship

[0167] Take the mixed reference solution after PMP derivatization in step 2.5.1 as the first concentration, and serially dilute it into a series of standard solutions. Inject continuously according to the chromatographic conditions of "2.5.2". Take the peak area as the ordinate and the concentration as the abscissa, plot the standard curve and perform linear regression.

[0168] 2.5.4 Methodology

[0169] Precision: Inject the mixed reference solution continuously for 6 injections, record the peak areas of each component and calculate the RSD%.

[0170] Repeatability: Take 6 portions of sample No. S10, derivatize them according to the method of "2.5.1", inject continuously, record the peak areas and calculate the RSD%.

[0171] Stability: Take 1 portion of the test sample and inject it at 0, 2, 4, 8, 12, and 24 h, record the peak areas of each monosaccharide, and calculate the RSD% value.

[0172] 3 Experimental results

[0173] 3.1 Determination results of neutral polysaccharide, uronic acid, and protein contents

[0174] Standard curves of the three detection methods. The regression equation of the glucose standard curve is y = 5.0283x + 0.0896, and the linear correlation coefficient R 2 = 0.9923; the regression equation of the glucuronic acid standard curve is y = 2.2054x + 0.0243, R 2 = 0.9921; the regression equation of the bovine serum albumin standard curve is: y = 18.563x + 0.5157, R 2= 0.9977. The results of the methodological investigation of the three detection methods are shown in Table 7. The precision RSD% of the three methods is 0.93% - 3.96%, indicating that the precision of the instruments used in the three methods is good. The repeatability RSD% of the three methods is 2.34% - 4.36%, indicating that the repeatability of the detection methods used is good. The stability RSD% of the three methods is 0.51% - 4.83%, indicating that the test samples are stable within 120 minutes. The average spiked recoveries of the three detection methods are 101.66% for the phenol-sulfuric acid method, RSD% = 2.49%, 99.32% for the m-hydroxybiphenyl method, RSD% = 2.25%, and 100.23% for the Coomassie brilliant blue method, RSD% = 3.01%, indicating that the three methods have good accuracy.

[0175] Table 7 Results of the methodological investigation

[0176]

[0177] The detection results are shown in Table 8. There are significant differences in the content of polysaccharide components in 13 batches of Isatis indigotica Fort. The content of neutral polysaccharide is between 55.36% - 79.38%. Among them, the highest sugar content is from Jiuquan, Gansu, which is 79.38%. The sugar contents of two batches of samples S8 and S9 from Daqing, Heilongjiang and Lindian, Heilongjiang are relatively low, which are 55.36% ± 0.31% and 58.69% ± 0.54% respectively. The uronic acid content of polysaccharides in 13 batches of Isatis indigotica Fort. is between 14.74% - 20.82%, and the difference is not significant. Among them, the content in Shanxi is the lowest, and the uronic acid content in Lemin, Gansu is the highest. The protein content of sample S9 is 2.74% ± 0.20%, which is the highest among all production areas.

[0178] Table 8 Determination results of neutral polysaccharide, acidic polysaccharide and protein contents in polysaccharides of 13 batches of Isatis indigotica Fort.

[0179] Number Neutral polysaccharide (%) Acidic polysaccharide (%) Protein (%) S1 75.14±2.04 18.82±0.62 0.94±0.03 S2 69.38±0.77 16.01±0.82 0.78±0.08 S3 62.11±0.92 14.74±0.52 1.72±0.08 S4 76.76±1.06 20.48±1.76 0.88±0.04 S5 72.00±0.14 20.12±1.20 0.66±0.01 S6 72.60±1.12 14.83±0.55 0.74±0.01 S7 76.57±2.49 16.02±0.89 0.37±0.03 S8 55.36±0.31 16.45±0.28 0.71±0.02 S9 58.69±0.54 18.07±0.45 2.74±0.20 S10 79.38±2.77 15.38±0.46 0.94±0.04 S11 64.94±0.61 20.82±1.47 1.69±0.02 S12 67.37±0.40 18.13±0.55 0.59±0.05 S13 74.88±1.39 16.68±0.18 0.64±0.06

[0180] 3.2 Determination results of monosaccharide content

[0181] The standard curves and linear ranges of each monosaccharide are shown in Table 9, and the separation effects of each monosaccharide are as Figure 5 . The results of the methodological investigation are shown in Table 10. The HPLC content determination method used in this experiment successfully separated 7 target monosaccharides, and had good linearity. The instrument precision, method reproducibility and stability of the test samples were all good. Substituting the peak areas of each chemical component of polysaccharides in each batch of Isatis indigotica Fort. into the standard curve to calculate the content results of each monosaccharide are shown in Table 11. Component 3, glucuronic acid, is its characteristic component. Among the 13 batches, polysaccharides from three batches of Isatis indigotica Fort., S3, S10 and S11, contain a small amount of glucuronic acid, while glucose has the highest content in all samples.

[0182] Table 9 Linear regression equations and linear ranges of 7 monosaccharides

[0183] Number Name Regression curve <![CDATA[R 2 > Linear range (μg / mL) 1 Man y = 262144x - 619433 0.9947 2.92～17.40 2 Rha y = 458583x - 674764 0.9946 1.32～10.03 3 Glc-A y = 194074x - 161843 0.9950 0.81～6.16 4 Gal-A y = 181683x - 188249 0.9972 1.08～8.30 5 Glc y = 121652.1730x - 1717745.7209 0.9972 34.10～258.93 6 Ara y = 133449x - 287798 0.9943 1.97～14.97 7 Gal y = 349778x - 677052 0.9951 1.71～13.01

[0184] Table 10 Results of methodological investigation

[0185] Precision (RSD / %) Repeatability (RSD / %) Stability (RSD / %) Man 0.67 1.82 1.23 Rha 1.24 4.76 4.34 Glc-A 0.93 2.68 1.52 Gal-A 0.79 3.98 2.35 Glc 1.17 2.63 2.56 Ara 0.65 3.79 4.84 Gal 0.23 4.83 4.47

[0186] Table 11 Determination results of monosaccharide contents in 13 batches of Isatis indigotica polysaccharide

[0187]

[0188] The above embodiments are only for illustrating the technical concept and characteristics of the present invention, and the purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it. However, the protection scope of the present invention cannot be limited thereby. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for detecting the quality of Radix Isatidis polysaccharides, characterized in that: The following steps are involved: (1) Establishment of UV spectrum: Take the Radix Isatidis polysaccharide sample and dissolve it in water to prepare a solution, use pure water as a blank, use a UV scanner to perform a full wavelength scan from 200 to 800 nm, and draw a UV spectrum; (2) Establishment of infrared spectrum: The Radix Isatidis polysaccharide sample was mixed with KBr in proportion and ground into slices, which were then scanned using a Fourier transform infrared spectrometer. The spectral range was mid-infrared 4000-400 cm -1 , draw infrared spectra; (3) Establishment of HPLC fingerprint of hydrolyzed monosaccharides: Weighing a Radix Isatidis polysaccharide sample into an ampoule, adding trifluoroacetic acid, and heating for hydrolysis to obtain hydrolyzed Radix Isatidis polysaccharide; Take the hydrolyzed Radix Isatidis polysaccharide, monosaccharide mixed solution, and monosaccharide solution, add NaOH solution, add 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution, mix well, put in a water bath, add HCl solution to neutralize the pH value after cooling, shake and add chloroform to extract, take the supernatant, filter with a 0.22 μm microporous membrane, set aside, perform HPLC analysis according to the set chromatographic conditions, and establish a fingerprint spectrum; (4) Establishment of HPGPC characteristic spectrum: Weigh the Radix Isatidis polysaccharide sample into a volumetric flask, dissolve it with sodium nitrate to prepare a polysaccharide solution, filter it through a 0.22 μm filter membrane, and collect the HPGPC spectrum according to the set high performance gel permeation chromatography method to generate a fingerprint spectrum; (5) Determination of neutral polysaccharide, uronic acid and protein content: Accurately weigh the Radix Isatidis polysaccharide sample, and determine the neutral polysaccharide, uronic acid and protein content using the sulfuric acid phenol method, the m-hydroxybiphenyl method and the Coomassie brilliant blue method respectively; (6) Determination of monosaccharide composition: The polysaccharide sample of Radix Isatidis after acid hydrolysis was subjected to PMP derivatization-HPLC analysis according to the set chromatographic conditions to determine the monosaccharide composition.

2. The method for detecting the quality of Radix Isatidis polysaccharides according to claim 1, characterized in that: Step (1) UV spectrum establishment: Different batches of Radix Isatidis polysaccharide samples were taken and dissolved in water to form solutions. Pure water was used as blank. A UV scanner was used to perform full wavelength scanning from 200 to 800 nm, and Origin 2022 software was used to draw the UV spectra of 13 batches of Radix Isatidis polysaccharides.

3. The method for quality detection of Radix Isatidis polysaccharides according to claim 1, characterized in that: Step (2) Infrared spectrum establishment: Different batches of Radix Isatidis polysaccharide samples were ground with KBr at a ratio of 1:100 and scanned with a Fourier transform infrared spectrometer in the mid-infrared range of 4000-400 cm -1 , Origin 2022 software was used to draw the infrared spectra of 13 batches of Isatis indigotica polysaccharides.

4. The method for quality detection of Radix Isatidis polysaccharides according to claim 1, characterized in that: Step (3) Establishment of HPLC fingerprint of hydrolyzed monosaccharides: The Radix Isatidis polysaccharide sample was put into an ampoule, trifluoroacetic acid was added, and the ampoule was sealed with an alcohol burner. The ampoule was acid-hydrolyzed in an oven at 110-120 ℃ for 4-6 h. Methanol was added and evaporated repeatedly until the trifluoroacetic acid was evaporated. Ultrapure water was added to re-dissolve. The hydrolyzed Radix Isatidis polysaccharide, monosaccharide mixed solution, and monosaccharide solution were added with NaOH solution and PMP methanol solution. The mixture was mixed and placed in a water bath. After cooling, HCl solution was added to neutralize the pH value. After shaking, chloroform was added to extract the mixture. The supernatant was taken and filtered with a 0.22 μm microporous filter membrane for standby use. HPLC analysis was performed according to the set chromatographic conditions to obtain the chromatogram, which was imported into the 2012 version of the "Chinese Medicine Chromatographic Fingerprint Similarity Detection System" software in CDF format to synthesize the fingerprint. S1 was used as the reference peak spectrum, and the reference spectrum was generated by the "average method". The time width was set to 0.1 min. Multi-point correction and Mark peak matching were performed on the chromatographic peaks of Radix Isatidis polysaccharides to establish the fingerprint.

5. The method for quality detection of Radix Isatidis polysaccharides according to claim 4, characterized in that: Step (3) The chromatographic conditions for HPLC analysis were: AlltimaTM C18 column, size 250 mm × 4.6 mm, 5 μm; mobile phase was 0.1 mol·L-1, pH 6.

8. -1 Phosphate buffer and acetonitrile, isocratic elution, volume ratio 83:17; column temperature 30 ℃; volume flow rate 1.0 mL·min -1 ; Injection volume 10 μL, detection wavelength 245 nm.

6. The method for quality detection of Radix Isatidis polysaccharides according to claim 1, characterized in that: The step of determining the similarity by the HPGPC method in step (4) comprises: taking a Radix Isatidis polysaccharide sample, dissolving it with sodium nitrate to prepare a Radix Isatidis polysaccharide solution of a certain concentration, collecting the HPGPC spectrum of the Radix Isatidis polysaccharide by high performance gel permeation chromatography after centrifugation and microporous membrane filtration, importing it into the "Chinese Medicine Chromatographic Fingerprint Similarity Detection System" software 2012 version in CDF format, synthesizing the fingerprint spectrum, and calculating the similarity of the Radix Isatidis polysaccharide.

7. The method for quality detection of Radix Isatidis polysaccharides according to claim 6, characterized in that: Step (4) The chromatographic conditions of the HPGPC characteristic spectrum are as follows: the detector is a Waters 2414 differential refractometer; the chromatographic column is a gel column TSK-gel G5000PW in series with TSK-gel G3000PW, with specifications of 8.0×300 mm, 6μM; Mobile phase: 0.1 mol·L -1 Sodium nitrate; flow rate: 0.4 mL min -1 ; Column temperature 35 ℃; Injection volume 10 μL.

8. The method for quality detection of Radix Isatidis polysaccharides according to claim 1, characterized in that: Step (5) The method for determining the content of neutral polysaccharides comprises the following steps: (1) Drawing of glucose standard curve Accurately weigh D-anhydrous glucose, make up to volume, accurately measure 0, 0.05, 0.1, 0.15, 0.2, 0.3 mL, respectively, put them into stoppered test tubes, add pure water to make up to 1.0 mL, add 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid and shake well, place in a 100℃ water bath for 20 minutes, and after cooling, measure the absorbance at a wavelength of 490 nm with A as the ordinate and glucose concentration C as the abscissa to draw a standard curve; (2) Sample measurement Accurately weigh Radix Isatidis polysaccharide in a volumetric flask, prepare the Radix Isatidis polysaccharide test solution with pure water, determine its absorbance value according to the method described in step (1), and substitute it into the standard curve to calculate the content; The uronic acid content determination includes the following steps (1) Solution preparation Sodium tetraborate-concentrated sulfuric acid solution: weigh sodium tetraborate, add concentrated sulfuric acid and fully dissolve; 0.15% m-hydroxybiphenyl solution: weigh m-hydroxybiphenyl into a volumetric flask, add 5% sodium hydroxide solution to dissolve to the mark; (2) Drawing of glucuronic acid standard curve Accurately weigh D-glucuronic acid, make up to volume, and obtain D-glucuronic acid standard solution. Accurately measure 0, 0.1, 0.2, 0.4, 0.6, and 0.7 mL of the standard solution, respectively, and place them in stoppered test tubes. Add pure water to make up to 1.0 mL, add 6 mL of sodium tetraborate-concentrated sulfuric acid and 0.1 mL of m-hydroxybiphenyl solution, shake well, and place at room temperature for 40 minutes. Measure the absorbance value A at 525 nm as the ordinate and the glucuronic acid concentration C as the abscissa to draw a standard curve. (3) Sample measurement Accurately weigh Radix Isatidis polysaccharide in a volumetric flask, prepare the Radix Isatidis polysaccharide test solution with pure water, make up with pure water, determine its absorbance value according to the method in step (2) above, and substitute it into the standard curve to calculate the content; Protein content determination (1) Solution preparation Test sample solution: Take Radix Isatidis polysaccharide in a volumetric flask, add pure water to dissolve it and then dilute to the mark to obtain Radix Isatidis polysaccharide test sample solution; Coomassie Brilliant Blue G250 solution: Accurately weigh Coomassie Brilliant Blue G250 powder into a volumetric flask, add ethanol, add H3PO4 solution, and make up to volume; (2) Plotting of protein standard curve Accurately weigh bovine serum albumin, add water to dissolve and make up to volume, accurately measure 0, 0.1, 0.2, 0.5, 0.6, and 0.7 mL of standard solution, respectively, place in stoppered test tubes, add pure water to make up to 1.0 mL, add 5 mL of Coomassie Brilliant Blue G250 solution, shake well, keep at room temperature for 5 minutes away from light, measure the absorbance value A at a wavelength of 595 nm on an ELISA reader as the ordinate, and draw a standard curve with the bovine serum albumin concentration C as the abscissa; (3) Sample measurement Take 1 mL of the Radix Isatidis polysaccharide test solution obtained in step (1), add 5 mL of Coomassie Brilliant Blue G250 solution, shake well, let stand at room temperature for 5 minutes, measure the absorbance at a wavelength of 595 nm on an ELISA reader, and substitute it into the standard curve in step (2) to calculate the content.

9. The method for quality detection of Radix Isatidis polysaccharides according to claim 1, characterized in that: Step (6) The monosaccharide content determination comprises the following steps: (1) Preparation of Radix Isatidis polysaccharide samples, monosaccharide solutions and mixed reference solutions Accurately weigh the Radix Isatidis polysaccharide sample into an ampoule and add 2 mol·L -1 The mixture was sealed with an alcohol burner, reacted in an oven at 110 °C for 4 h, and the trifluoroacetic acid was removed by rotary evaporation. Methanol was added for re-dissolution, and the trifluoroacetic acid was evaporated to dryness. Ultrapure water was added for re-dissolution to obtain a Radix Isatidis polysaccharide sample solution. Accurately weigh mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, arabinose, and galactose, and prepare a mixed reference solution of a certain concentration with ultrapure water for later use; Take 500 μL of the mixed reference solution and Radix Isatidis polysaccharide sample solution, add 0.3 mol·L -1 75 μL of NaOH solution was added, and then 150 μL of 10% PMP methanol solution was added, vortexed and mixed, and placed in a 70℃ water bath for 1 h. After cooling to room temperature, 0.3 mol·L -1 75 μL of HCl solution was added to neutralize the pH, and after shaking, chloroform was added to extract three times, and the supernatant was filtered through a 0.22 μm microporous membrane; (2) Linear relationship investigation Take the mixed reference solution after PMP derivatization in step (1) as the first concentration, dilute it step by step into a series of standard solutions, inject samples continuously, use the peak area as the ordinate and the concentration as the abscissa, draw a standard curve and perform linear regression; (3) Determination of monosaccharide content Take the Radix Isatidis polysaccharide sample in step (1) and inject it according to the chromatographic conditions in step (2), and substitute it into the standard curve in step (2) to calculate the monosaccharide content in the Radix Isatidis polysaccharide.

10. The method for quality detection of Radix Isatidis polysaccharides according to claim 9, characterized in that: Step (2) Chromatographic conditions: AlltimaTM C18 column, size 250 mm × 4.6 mm, 5 μm; mobile phase: 0.1 mol·L-1, pH 6.8 -1 Phosphate buffer-acetonitrile, isocratic elution, volume ratio of 83:17; column temperature 30 ℃; volume flow rate 1.0 mL·min -1 ; Injection volume 10 μL, detection wavelength 245 nm.

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