A method for establishing a multivariate fingerprint of Polygonatum sibiricum origin species

Abstract

The present invention provides a method for establishing a multivariate fingerprint of a Polygonatum sibiricum original species. The method for establishing the fingerprint comprises: (1) preparing a reference solution; (2) preparing a test solution; and (3) establishing the fingerprint. The method of the present invention combines the fingerprint of HILIC-HPLC-ELSD hydrolysate to establish a multiple fingerprint for evaluating the quality of Polygonatum sibiricum polysaccharide (PCP). Similarity evaluation was performed on 34 batches of Polygonatum sibiricum polysaccharides from different sources. Chemometric methods, including HCA, PCA and PLS-DA, were used to fuse, process and analyze the chromatographic data of multiple PCPs. The method of the present invention has good separation, good peak shape and high resolution.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and in particular to a method for establishing a multivariate fingerprint spectrum of polygonatum-based species. Background Art

[0002] The 2015 edition of the Chinese Pharmacopoeia, Volume 1, specifies the source of Polygonatum sibiricum as follows: "This product is the dried rhizome of Polygonatum kingianum Coll. et Hemsl., Polygonatum sibiricum Red., or Polygonatum cyrtonema Hua, all of the Liliaceae family." Depending on its shape, it is commonly known as "big polygonatum," "chicken-headed polygonatum," or "ginger-shaped polygonatum." Recognized by the National Health and Family Planning Commission as a traditional Chinese medicinal herb that functions as both a food and a medicinal ingredient, it boasts benefits such as tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. It is commonly used for fatigue relief, immune regulation, and lowering blood sugar and lipids, possessing exceptionally high medicinal and health benefits. Due to its proven efficacy, strong market recognition, and high price, other species of the genus Polygonatum, such as Hubei and Sichuan polygonatum, are often sold as counterfeit, severely impacting the development of the polygonatum industry. An important reason why this counterfeiting phenomenon is difficult to eliminate is that the medicinal material standards of Polygonatum sibiricum are not clear enough and the method specificity is poor. It is difficult to quickly evaluate the quality of Polygonatum sibiricum from different sources through the existing medicinal material standards. Therefore, there is an urgent need to conduct research on the technical standards of Polygonatum sibiricum Chinese medicinal materials to form clearer and more distinctive standards.

[0003] Furthermore, research has found that Polygonatum sibiricum contains a rich variety of sugars, but their distribution is uneven. As the primary chemical component of Polygonatum sibiricum, measuring their content and determining their purity are essential tools for evaluating the quality of Polygonatum sibiricum polysaccharides. However, the commonly used phenol-sulfuric acid method suffers from low accuracy and inability to obtain structural information about the polysaccharides, making it difficult to effectively assess the quality of Polygonatum sibiricum polysaccharides from different commercial sources. While partial acid hydrolysis is currently a simple and easy method widely used for the degradation of polysaccharides in traditional Chinese medicine, the products of partial acid hydrolysis are generally more complex and less uniform than those produced by enzymatic hydrolysis. Enzymatic hydrolysis offers mild conditions and high specificity.

[0004] For example, in the reference, the title is "Analysis of polysaccharide differences of the original species of Polygonatum sibiricum based on PMP-HPLC and chemometrics". This method uses the water extraction and alcohol precipitation method to extract the polysaccharides in the medicinal material of Polygonatum sibiricum. After trifluoroacetic acid (TFA) hydrolysis and 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization, HPLC is used to establish the chromatographic fingerprint of the three species of Polygonatum sibiricum. The fingerprints are analyzed by similarity (SA) analysis, cluster analysis (HCA) and principal component analysis (PCA) to study the differences of the polysaccharides of the three original species of Polygonatum sibiricum. However, this method has the following defects: (1) It uses The 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization agent undergoes a pre-column derivatization reaction, which will cause excessive material components, have a negative impact on the column and detection, and have poor precision and controllability; (2) The sample preparation process of this method is relatively complex, time-consuming, and costly; (3) From the cluster analysis of polysaccharide chromatographic fingerprints in this study, the polysaccharide components of Polygonatum sibiricum, Polygonatum sibiricum and Polygonatum sibiricum are significantly different, while the polysaccharide fingerprints of Polygonatum sibiricum and Polygonatum sibiricum are slightly different from those of Polygonatum sibiricum, and the chromatographic fingerprints between the two are slightly different from those of Polygonatum sibiricum, and they cannot be distinguished by cluster analysis; this is inconsistent with the conclusion of principal component analysis; the reliability of this method is poor.

[0005] Therefore, the study of Chinese herbal polysaccharides based on enzymatic hydrolysis is more directional, purposeful, and controllable. In this study, the enzymatic hydrolysis products of Polygonatum sibiricum polysaccharides were further studied using HPLC-HILIC-ELSD. In addition, the HILIC mode was combined with ELSD technology to separate and detect oligosaccharides. As a result, all peaks were separated, with good peak shape and high resolution. The chromatographic data of various Polygonatum sibiricum polysaccharides (PCPs) were fused, processed, and analyzed using chemometric methods, including HCA (hierarchical cluster analysis), PCA (principal component analysis), and PLS-DA (partial least squares discriminant analysis), verifying the accuracy and reliability of this method. Summary of the Invention

[0006] In response to the deficiencies in the prior art, the present invention provides a method for establishing a multivariate fingerprint of a polygonatum-based species and for chemical pattern recognition, which is simple to operate, accurate, and reliable; an enzymatic method is used to hydrolyze polygonatum polysaccharides, a fructosidase suitable for polygonatum is screened, and the enzymatic hydrolysis conditions and methods are optimized; specific oligosaccharide fragments in polygonatum polysaccharides are detected, the enzymatic hydrolysis conditions are mild and specific, and the hydrolysis reaction is more directional, purposeful, and controllable; an evaporative light detector that is more suitable for separating oligosaccharide fragments in polysaccharides is used, and combined with a HILIC chromatographic column, the final polygonatum polysaccharide fingerprint can reach 17 chromatographic peaks; a fingerprint of polygonatum polysaccharides is constructed, and the fingerprint of the present invention is stable and reliable after verification by methodological and chemometric methods; and to a certain extent, the problem of identifying easily confused polygonatum products such as Sichuan polygonatum and Hubei polygonatum in the current market is solved.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for establishing a multivariate fingerprint of a Polygonatum basilicum species comprises the following steps:

[0009] (1) Preparation of reference solution: Take appropriate amount of fructose, sucrose, and glucose reference substances, accurately weigh them, transfer them to a volumetric flask, and add water to the mark.

[0010] (2) Preparation of test solution: Take an appropriate amount of Polygonatum sibiricum polysaccharide and mix it with fructosidase solution, place it in an oscillator for hydrolysis, heat it, centrifuge it, take the supernatant, dry it, and dissolve it in acetonitrile / water solution to obtain the product;

[0011] (3) Establishment of fingerprint: Use high performance liquid chromatography to detect the reference solution and the test solution, record the chromatogram, and establish the fingerprint of the reference solution and the test solution; the chromatographic conditions are as follows:

[0012] Chromatographic column: Hypersil GOLD TM PEI HILIC HPLC, model: 5 μm, 250 mm × 4.6 mm; mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution; column temperature: 35 °C; flow rate: 1.0 mL min -1 ; Detector: evaporative light detection; injection volume 10 μL;

[0013] Gradient elution conditions: 0-25 min, 85%-65% A; 25-35 min, 65%-50% A.

[0014] The preparation of the reference solution in step (1) of the present invention is as follows: 10 mg each of fructose, sucrose and glucose reference substances are accurately weighed, transferred to a 10 mL volumetric flask, and diluted to the mark with water.

[0015] The preparation of the test solution described in step (2) of the present invention is as follows: take Polygonatum sibiricum polysaccharide, add water to make a Polygonatum sibiricum polysaccharide solution with a concentration of 0.5 mg / mL, add an equal volume of fructosidase solution and mix, place in an oscillator for hydrolysis, heat, centrifuge, take the supernatant, dry, and dissolve with 500 μL of acetonitrile / water solution to obtain.

[0016] The concentration of the fructosidase solution in step (2) of the present invention is 50-200 U / mL.

[0017] Preferably, the concentration of the fructosidase solution in step (2) of the present invention is 100 U / mL.

[0018] The heating in step (2) of the present invention is: heating at 80° C. for 20 minutes.

[0019] The setting conditions of the oscillator in step (2) of the present invention are: temperature of 55° C., rotation speed of 200 rpm, and time of 1-7 hours.

[0020] Preferably, the setting conditions of the oscillator in step (2) of the present invention are: temperature of 55° C., rotation speed of 200 rpm, and time of 3 hours.

[0021] The centrifugal setting conditions in step (2) of the present invention are: 4500r / min, 15min.

[0022] The acetonitrile / water solution in step (2) of the present invention has a volume ratio of 1:1.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention adopts an enzymatic hydrolysis method to hydrolyze polygonatum polysaccharide, screens out fructosidase suitable for polygonatum, and optimizes the enzymatic hydrolysis conditions and methods. When the enzyme concentration is 100 U / mL, there are more chromatographic peaks of oligosaccharides with higher polymerization degree; when the hydrolysis time is 3 h, the number and area of ​​the peaks are relatively the best; finally, the polygonatum polysaccharide fingerprint can reach 17 chromatographic peaks, and sugar components with a polymerization degree of 7-8 can be detected. The efficient and accurate research system plays an important role in further promoting the research, application and rapid development of polygonatum polysaccharide.

[0025] 2. Specific oligosaccharide fragments in Polygonatum sibiricum polysaccharide are separated and detected by glycosidase enzymatic hydrolysis. The enzymatic hydrolysis conditions are mild and the specificity is strong. The hydrolysis reaction is more directional, purposeful and controllable, which has significant advantages in analyzing the glycosidic bond type of polysaccharides, constructing sugar spectra and studying structure-activity relationships. It solves the problems of the phenol-sulfuric acid method commonly used in the existing technology, such as that some acid hydrolysis products are generally complex, have poor uniformity, low accuracy and cannot obtain structural information of polysaccharides, and cannot effectively evaluate the quality of Polygonatum sibiricum polysaccharides from different sources.

[0026] 3. The present invention adopts a method combining a sensitive evaporative light detector with a HILIC chromatographic column to separate and detect oligosaccharides without pre-column derivatization. The results show that all peaks are separated, the peak properties are good, the resolution is high, and the specificity is good, which solves the problem of poor specificity of existing Polygonatum quality detection methods.

[0027] 4. The present invention conducted a methodological investigation on the established fingerprint spectrum, and the results were as follows: (1) Through the precision test, the RSD of the relative retention time and relative peak area of ​​the common peaks were measured to be less than 3%, indicating that the instrument precision was good and met the requirements of the fingerprint spectrum; (2) Through the repeatability test, the RSD of the relative retention time and relative peak area of ​​each common peak was measured to be less than 3%, and the results of each extraction of the same sample were relatively consistent, indicating that the extraction method had good reproducibility and met the requirements of the fingerprint spectrum; (3) Through the stability test, the RSD of the relative retention time and relative peak area of ​​the common peaks was measured to be less than 3%, indicating that the test solution was relatively stable within 24 hours, and the chemical composition and content would not change, which met the requirements of the fingerprint spectrum; In summary, it is shown that the method of the present invention is stable and feasible.

[0028] 5. The method of the present invention uses fingerprint similarity evaluation. 17 peaks corresponding to enzymatic hydrolysis products are detected by HPLC-HILIC-ELSD. By comparing the retention times of fructose, glucose and sucrose under the same conditions, the DPs of peaks 1 to 7 are preliminarily determined. The chromatograms of 5 batches of Polygonatum sibiricum, 5 batches of Polygonatum duniflorum, 10 batches of Polygonatum sibiricum, 5 batches of Polygonatum sibiricum from Sichuan, 5 batches of Polygonatum sibiricum from Hubei, 2 batches of Polygonatum sibiricum from small leaves and 2 batches of imported Polygonatum sibiricum are respectively imported into the Chinese medicine fingerprint similarity evaluation system (version 2.0) software to generate reference spectra of 3 kinds of original Polygonatum sibiricum. The 34 batches of samples are compared with the reference spectra of Polygonatum sibiricum, Polygonatum duniflorum and Polygonatum duniflorum, and the similarity is calculated. The results are as follows:

[0029] ①With Polygonatum multiflorum Compared with the reference map, the similarity of 10 batches of Polygonatum multiflorum among the three original Polygonatum sibiricum is greater than 0.93. Compared with them, the similarity of Polygonatum yunnanensis and Polygonatum acanthopanax is less than 0.85, except for one batch of Polygonatum yunnanensis (D1). Compared with the reference map of Polygonatum multiflorum, the distribution of easily confused products on the market is more discrete, among which the similarity of Hubei Polygonatum is between 0.44 and 0.95; the similarity of Sichuan Polygonatum is less than 0.86 except for one batch (C4); the similarity of imported Polygonatum is less than 0.82; the similarity of Polygonatum microphylla is less than 0.5;

[0030] ②With Polygonatum dahliae Comparison of the chromatograms of the three original Polygonatum sibiricum samples revealed similarities of >0.90 for all five batches of Yunnan Polygonatum sibiricum. The similarities of Polygonatum sibiricum and Polygonatum multiflorum to the Yunnan Polygonatum sibiricum chromatograms were <0.90, with the exception of Polygonatum multiflorum (S1, S8, S9, S10) and Polygonatum sibiricum (J2). The similarities of other commercially available Polygonatum sibiricum samples ranged from 0.65 to 0.99, with Sichuan Polygonatum sibiricum having similarities >0.90. Some similarities between Hubei Polygonatum and imported Polygonatum sibiricum were >0.90, while those of Polygonatum microphylla were <0.80.

[0031] ③With Polygonatum odoratumComparison of the chromatograms revealed that the similarities of the three ancestral Polygonatum species were >0.99 for all five batches of Polygonatum sibiricum. The similarities of Polygonatum yunnanensis and Polygonatum multiflorum, with the exception of Polygonatum multiflorum (S7) and Polygonatum yunnanensis (D4), were <0.80. The similarities of other commercially available Polygonatum species ranged from 0.60 to 0.99, with the similarities of Polygonatum sichuanensis all <0.80; the similarities of Polygonatum sichuanensis with the exception of H5 were <0.70; and the similarities of Polygonatum sibiricum with the exception of imported Polygonatum sibiricum (Z2) were >0.90.

[0032] These results indicate that the fingerprint chromatograms within the three different sources of Polygonatum sibiricum listed in the Pharmacopoeia are highly similar, with minimal differences. The three original Polygonatum sibiricum species exhibit significant interspecific differences, while some easily confused products on the market also exhibit slight differences from the three Polygonatum sibiricum species listed in the Pharmacopoeia. Therefore, the structural characteristics of different Polygonatum sibiricum polysaccharides are distinct, making them easier to distinguish.

[0033] 6. The present invention has, but is not limited to, research on varieties of polygonatum included in the pharmacopoeia in terms of sample collection, which to a certain extent solves the problem of identifying easily confused polygonatum products such as Sichuan polygonatum and Hubei polygonatum in the current market.

[0034] 7. The chemometric analysis results of the present invention for the Polygonatum varieties included in the pharmacopoeia are as follows:

[0035] (1) Cluster analysis: 20 batches of Polygonatum sibiricum samples were divided into three categories: the first category included 10 batches of Polygonatum cyrtonema, which were collected from Jinzhai and Shitai (3) in Anhui Province, Hangzhou (2), Chun'an (2), Quzhou in Zhejiang Province, and Ruichang in Jiangxi Province; the second category included 5 batches of Polygonatum yunnanensis, which were collected from Dazhou, Chengdu (2) in Sichuan Province, Wuhan in Hubei Province, Kunming in Yunnan Province, and Zhijin in Guizhou Province; the third category included 5 batches of Polygonatum acutiloba, which were collected from Dafang in Guizhou Province, Chizhou in Anhui Province, Zaozhuang in Shandong Province, and Qingyuan in Liaoning Province;

[0036] (2) Principal component analysis: The peak areas of 17 common peaks of 20 batches of samples were imported into SPSS 23.0 software for PCA analysis. With the eigenvalue > 1 as the extraction standard, four principal components were extracted. The first three PCs accounted for 50.1%, 26.6% and 9.1% of the total variance, respectively. The three principal components represented 85.8% of the information content of the 17 components in Polygonatum sibiricum polysaccharide, which was sufficient to evaluate the quality of Polygonatum sibiricum polysaccharide. The data matrix was imported into SIMCA statistical software and three principal components were extracted. In this model, R 2 X(cum)=0.844,Q 2 (cum) = 0.612, all greater than 0.5, indicating that the PCA model has better predictive ability. The three-dimensional PCA score plot of PCP shows that the 20 batches of PCP are divided into three categories, which is consistent with the HCA results; Polygonatum multiflorum S1-S10 are clustered into one group, Polygonatum yunnanensis S11-S15 are clustered into another group, and Polygonatum acanthopanax S16-S20 are classified into the third group;

[0037] (3) Partial least squares discriminant analysis: The data matrix was imported into SIMCA statistical software and analyzed by PLS-DA. In this model, R 2 X(cum)=0.909,R 2 Y(cum)=0.863,Q 2 (cum) = 0.775, R 2 Y and Q 2 The value of is preferably greater than 0.5, and R 2 Y and Q 2 The closer the value of is to 1, the better the prediction ability of the PLS-DA model is. Figure 12 As shown, the classification results are consistent with those of HCA and PCA. Figure 13 It reflects the VIP value of the model. The size of the VIP value indicates the contribution of each independent variable. Taking VIP>1 as the screening standard, the characteristic components that cause differences between origins are obtained; from the VIP value, it can be seen that the 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 above three analysis methods verify that the enzymatic hydrolysis method is stable, accurate and feasible for analyzing Polygonatum sibiricum polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the chromatogram showing the effect of enzyme concentration of 10 U / mL on PCP hydrolysis;

[0039] Figure 2 This is the chromatogram showing the effect of enzyme concentration of 50 U / mL on PCP hydrolysis;

[0040] Figure 3 The chromatogram shows the effect of enzyme concentration of 100 U / mL on PCP hydrolysis;

[0041] Figure 4 The chromatogram shows the effect of enzyme concentration of 200 U / mL on PCP hydrolysis;

[0042] Figure 5 This is the chromatogram showing the effect of 1 h reaction time on PCP hydrolysis;

[0043] Figure 6 This is the chromatogram showing the effect of 3 h reaction time on PCP hydrolysis;

[0044] Figure 7 This is the chromatogram showing the effect of 5 h reaction time on PCP hydrolysis;

[0045] Figure 8 This is the chromatogram showing the effect of 7 h reaction time on PCP hydrolysis;

[0046] Figure 9 This is the chromatogram showing the effect of 5 mg sampling on PCP hydrolysis;

[0047] Figure 10 The chromatogram shows the effect of 10 mg sampling volume on PCP hydrolysis;

[0048] Figure 11 This is a diagram showing the separation effect of the Sunniest HILIC-S column;

[0049] Figure 12 Investigating the separation effect of the Welch Ultimate HILIC Amide column

[0050] Figure 13 Hypersil GOLD column TM PEI HILIC HPLC separation effect diagram

[0051] Figure 14 This is the chromatogram showing the effect of column temperature at 30°C on PCP enzymatic hydrolysis;

[0052] Figure 15 This is the chromatogram showing the effect of column temperature at 35°C on PCP enzymatic hydrolysis;

[0053] Figure 16 This is the chromatogram showing the effect of column temperature at 40°C on PCP enzymatic hydrolysis;

[0054] Figure 17 This is the chromatogram of the separation effect of the mobile phase methanol-water on the sample;

[0055] Figure 18 This is the chromatogram of the separation effect of the mobile phase acetonitrile-water on the sample;

[0056] Figure 19 This is the chromatogram showing the effect of evaporative light drift tube temperature at 90°C on PCP enzymatic hydrolysis;

[0057] Figure 20 This is the chromatogram of the effect of evaporative light drift tube temperature of 100℃ on PCP enzymatic hydrolysis;

[0058] Figure 21 This is the chromatogram showing the effect of evaporative light drift tube temperature of 105°C on PCP enzymatic hydrolysis;

[0059] Figure 22 The chromatogram shows the effect of 5 μL injection volume on PCP hydrolysis;

[0060] Figure 23 The chromatogram shows the effect of 10 μL injection volume on PCP hydrolysis;

[0061] Figure 24 The chromatogram shows the effect of 15 μL injection volume on PCP hydrolysis;

[0062] Figure 25The chromatograms of Polygonatum sibiricum sample and reference substance are shown;

[0063] Figure 26 is the chromatogram of fructose reference substance;

[0064] Figure 27 is the chromatogram of glucose reference;

[0065] Figure 28 is the chromatogram of sucrose reference substance;

[0066] Figure 29 The fingerprints of 10 batches of Polygonatum cyrtonema (S1-S10 are Polygonatum cyrtonema)

[0067] Figure 30 The fingerprints of Polygonatum sibiricum, Polygonatum sibiricum and Polygonatum sibiricum are shown in Figure 1. (D1-D5 are Polygonatum sibiricum; J1-J5 are Polygonatum sibiricum; H1-H5 are Polygonatum sibiricum)

[0068] Figure 31 Fingerprints of Polygonatum sibiricum, Sichuan Polygonatum sibiricum, and imported Polygonatum sibiricum; (C1-C5 are Sichuan Polygonatum sibiricum; Z1-Z2 are imported Polygonatum sibiricum; X1-X2 are Polygonatum sibiricum)

[0069] Figure 32 This is the HCA tree relationship diagram of 20 batches of Polygonatum sibiricum samples; (1-10 are Polygonatum sibiricum; 11-15 are Polygonatum sibiricum; 16-20 are Polygonatum sibiricum)

[0070] Figure 33 This is the gravel map of 20 batches of Polygonatum sibiricum samples;

[0071] Figure 34 This is the three-dimensional projection diagram of principal component analysis of 20 batches of Polygonatum sibiricum samples;

[0072] Figure 35 This is the partial least squares discriminant analysis diagram of 20 batches of Polygonatum sibiricum samples;

[0073] Figure 36 VIP images of 20 batches of Polygonatum sibiricum samples. DETAILED DESCRIPTION

[0074] The technical solution of the present invention is further described in detail below through specific embodiments.

[0075] Example 1 Method for Establishing Multivariate Fingerprints of Polygonatum sibiricum Species

[0076] (1) Preparation of reference solution: Take 10 mg each of fructose, sucrose, and glucose reference solutions, accurately weigh them, transfer them to a 10 mL volumetric flask, and dilute to the mark with water.

[0077] (2) Preparation of test solution: 5 mg of Polygonatum sibiricum polysaccharide was added to 10 mL of water to prepare a Polygonatum sibiricum polysaccharide solution with a concentration of 0.5 mg / mL. Then, 2.5 mL of the Polygonatum sibiricum polysaccharide solution was mixed with 2.5 mL of fructosidase solution (100 U / mL). The mixture was hydrolyzed in an oscillator (55°C, 200 rpm, 3 hours) and heated at 80°C for 20 minutes to denature the enzyme. The mixture was centrifuged (4500 rpm, 15 minutes), the supernatant was collected, dried, and dissolved in 500 μL of acetonitrile / water (1:1, v / v) solution to obtain the product.

[0078] (3) Establishment of fingerprint: Use high performance liquid chromatography to detect the reference solution and the test solution, record the chromatogram, and establish the fingerprint of the reference solution and the test solution; the chromatographic conditions are as follows:

[0079] Chromatographic column: Hypersil GOLD TM PEI HILIC HPLC, model: 5 μm, 250 mm × 4.6 mm; mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution; column temperature: 35 °C; flow rate: 1.0 mL min -1 ; Detector: evaporative light detection; injection volume 10 μL;

[0080] Gradient elution conditions: 0-25 min, 85%-65% A; 25-35 min, 65%-50% A.

[0081] Example 2 Method for Establishing Multivariate Fingerprints of Polygonatum sibiricum Species

[0082] (1) Preparation of reference solution: Take 10 mg of fructose, sucrose, and glucose reference solution, accurately weigh them, transfer them to a 10 mL volumetric flask, and dilute to the mark with water.

[0083] (2) Preparation of test solution: 2.5 mL of the Polygonatum sibiricum polysaccharide solution prepared in Example 1 was mixed with 2.5 mL of fructosidase solution (50 U / mL); the mixture was hydrolyzed in an oscillator (55°C, 200 rpm, 1 hour) and heated at 80°C for 20 minutes to denature the enzyme; the mixture was centrifuged (4500 rpm, 15 minutes), the supernatant was collected, dried, and dissolved in 500 μL of acetonitrile / water (1:1, v / v) solution to obtain the product;

[0084] (3) Establishment of fingerprint: Use high performance liquid chromatography to detect the reference solution and the test solution, record the chromatogram, and establish the fingerprint of the reference solution and the test solution; the chromatographic conditions are as follows:

[0085] Chromatographic column: Hypersil GOLD TMPEI HILIC HPLC, model: 5 μm, 250 mm × 4.6 mm; mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution; column temperature: 35 °C; flow rate: 1.0 mL min -1 ; Detector: evaporative light detection; injection volume 10 μL;

[0086] Gradient elution conditions: 0-25 min, 85%-65% A; 25-35 min, 65%-50% A.

[0087] Example 3 Method for Establishing Multivariate Fingerprints of Polygonatum Origin Species

[0088] (1) Preparation of reference solution: Take 10 mg of fructose, sucrose, and glucose reference solution, accurately weigh them, transfer them to a 10 mL volumetric flask, and dilute to the mark with water.

[0089] (2) Preparation of test solution: 2.5 mL of the Polygonatum sibiricum polysaccharide solution prepared in Example 1 was mixed with 2.5 mL of fructosidase solution (200 U / mL); the mixture was hydrolyzed in an oscillator (55°C, 200 rpm, 7 hours) and heated at 80°C for 20 minutes to denature the enzyme; the mixture was centrifuged (4500 rpm, 15 minutes), the supernatant was collected, dried, and dissolved in 500 μL of acetonitrile / water (1:1, v / v) solution to obtain the product;

[0090] (3) Establishment of fingerprint: Use high performance liquid chromatography to detect the reference solution and the test solution, record the chromatogram, and establish the fingerprint of the reference solution and the test solution; the chromatographic conditions are as follows:

[0091] Chromatographic column: Hypersil GOLD TM PEI HILIC HPLC, model: 5 μm, 250 mm × 4.6 mm; mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution; column temperature: 35 °C; flow rate: 1.0 mL min -1 ; Detector: evaporative light detection; injection volume 10 μL;

[0092] Gradient elution conditions: 0-25 min, 85%-65% A; 25-35 min, 65%-50% A.

[0093] Example 4 Method for Establishing Multivariate Fingerprints of Polygonatum sibiricum Species

[0094] (1) Preparation of reference solution: Take 10 mg of fructose, sucrose, and glucose reference solution, accurately weigh them, transfer them to a 10 mL volumetric flask, and dilute to the mark with water.

[0095] (2) Preparation of test solution: 2.5 mL of the polygonatum polysaccharide solution prepared in Example 1 was mixed with 2.5 mL of fructosidase solution (100 U / mL); the mixture was hydrolyzed in an oscillator (55°C, 200 rpm, 5 hours) and heated at 80°C for 20 minutes to denature the enzyme; the mixture was centrifuged (4500 rpm, 15 minutes), the supernatant was collected, dried, and dissolved in 500 μL of acetonitrile / water (1:1, v / v) solution to obtain the product;

[0096] (3) Establishment of fingerprint: Use high performance liquid chromatography to detect the reference solution and the test solution, record the chromatogram, and establish the fingerprint of the reference solution and the test solution; the chromatographic conditions are as follows:

[0097] Chromatographic column: Hypersil GOLD TM PEI HILIC HPLC, model: 5 μm, 250 mm × 4.6 mm; mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution; column temperature: 35 °C; flow rate: 1.0 mL min -1 ; Detector: evaporative light detection; injection volume 10 μL;

[0098] Gradient elution conditions: 0-25 min, 85%-65% A; 25-35 min, 65%-50% A.

[0099] In order to further verify the feasibility of the present invention, the inventors conducted a series of experiments, as follows:

[0100] 1. Exploratory test of the method for establishing fingerprint spectrum

[0101] 1. Instruments and reagents

[0102] 1.1 Instrument

[0103] U3000 high performance liquid chromatograph (equipped with Allech ELSD6000 evaporative light scattering detector Dionex);

[0104] ML204 1 / 10,000 electronic balance, XP26 1 / 100,000 electronic balance, Toledo Instruments Shanghai Co., Ltd.;

[0105] Elmasonic S 30(H) ultrasonic cleaner, Tegent Technology Co., Ltd.;

[0106] Advantage A10 Milli-Q ultrapure water instrument, Murray (Shanghai) Biotechnology Co., Ltd.

[0107] 1.2 Drug testing

[0108] There are 34 batches of samples in total, which are samples of easily confused products in the market. The specific sources are shown in Table 1.

[0109] Table 1 Detailed list of test sample materials

[0110]

[0111] Glycosidase: Sucrase (Shanghai Yuanye Biotechnology); Fructosidase specification is 100U / Mg.

[0112] Reference substances and other reagents: Fructose reference substance (batch number 111504-201703, purity 99.8%, provided by the China Food and Drug Administration), sucrose reference substance (batch number 111507-201303, purity 99.8%, provided by the China Food and Drug Administration); glucose reference substance (Sigma). All reagents used in the experiment were of chromatographic grade, and all other reagents were of analytical grade; water was purified.

[0113] 2 Experimental methods

[0114] 2.1 Investigation of test sample preparation methods

[0115] This study pre-established a common chromatographic condition for Polygonatum sibiricum from different sources when hydrolyzed by different glycosidases, that is, under this condition, the main chromatographic peaks after hydrolysis by different glycosidases can be well separated.

[0116] 2.1.1 Investigation of glycosidase concentration

[0117] Dilute fructosidase with water to an enzyme solution of appropriate concentration, i.e. 10U / mL, 50U / mL,

[0118] 100 U / mL and 200 U / mL, where:

[0119] 10U / ml: For a volume of 10ml, 100U of enzyme is required, i.e. 1mg of fructosidase is taken;

[0120] 50U / ml: For a volume of 10ml, 500U of enzyme is needed, i.e. 5mg of fructosidase is taken;

[0121] 100U / ml: For a volume of 10ml, 1000U of enzyme is needed, i.e. 10mg of fructosidase is taken;

[0122] 200U / ml: The volume is 10ml, and 2000U of enzyme is needed, that is, 20mg of fructosidase is taken.

[0123] Fructosidase was diluted with water to prepare enzyme solutions of appropriate concentrations, namely 10 U / mL, 50 U / mL, 100 U / mL, and 200 U / mL. 5 mg of PCPs was accurately weighed into 10 mL centrifuge tubes, four parts in parallel, and then mixed with different enzyme solutions (2.5 mL). The mixture was shaken three times in an oscillator (55°C, 200 r / min, 3 h) for hydrolysis. The mixture was then heated at 80°C for 20 minutes to denature the enzyme. After centrifugation (4500 r / min, 15 min), the supernatant was collected, dried, dissolved in 500 μL of acetonitrile / water (1:1, v / v) solution, and further tested in HILIC mode.

[0124] Effects of different enzyme concentrations on hydrolysis Figures 1-4 As shown in the figure, at an enzyme concentration of 10 U / mL, there are almost no oligosaccharide peaks. At an enzyme concentration of 50 U / mL, the response value of the oligosaccharide peak is very low. At an enzyme concentration of 100 U / mL, there are more chromatographic peaks for oligosaccharides with higher polymerization degrees compared to an enzyme concentration of 200 U / mL. To obtain more oligosaccharide information, 100 U / mL was selected as the optimal enzyme concentration.

[0125] 2.1.2 Investigation of enzymatic hydrolysis methods

[0126] A PCP solution (5 mg) was mixed with fructosidase (100 U / mL, 2.5 mL) in four parallel portions. The mixture was shaken in a shaker (55°C, 200 rpm) for 1, 3, 5, and 7 hours. The mixture was then heated at 80°C for 20 minutes to denature the enzyme. After centrifugation (4500 rpm, 15 minutes), the supernatant was collected, dried, and dissolved in 500 μL of acetonitrile:water (1:1, v / v).

[0127] Effect of reaction time on hydrolysis Figure 5-Figure 8 As shown, with increasing reaction time, oligosaccharides with higher polymerization degrees are further hydrolyzed to oligosaccharides with lower polymerization degrees. Compared with the 1-h hydrolyzate, the chromatogram of the 3-h hydrolyzate shows increased areas of two peaks with retention times of 15 to 25 minutes, while the peak with retention times of 27 to 40 minutes increases. These results indicate that a 3-h hydrolysis time is more complete than a 1-h hydrolysis time. However, as the hydrolysis time continues to increase, the number and area of ​​the peaks do not change significantly. Therefore, considering both hydrolysis efficiency and effectiveness, 3 h was selected as the optimal hydrolysis time.

[0128] 2.1.3 Investigation of sampling volume

[0129] The mixing problem of polysaccharide solution and glycosidase solution was involved. The final volume of the test sample was set to 2mL. The sample weights of 5mg and 10mg were examined respectively. The results were compared as follows: Figure 9-10, it was found that when the sample weight continued to increase, the number and area of ​​the peaks did not change significantly. Therefore, considering the experimental efficiency and effect comprehensively, 10 mg was selected as the optimal sampling amount.

[0130] 2.1.4 Determination of test sample preparation method

[0131] Test solution preparation: Mix 5 mg of PCP with 2.5 mL of fructosidase solution (100 U / mL). The mixture is hydrolyzed in an oscillator (55°C, 200 rpm, 3 hours) and heated at 80°C for 20 minutes to denature the enzyme. After centrifugation (4500 rpm, 15 minutes), the supernatant is collected, dried, and dissolved in 500 μL of acetonitrile / water (1:1, v / v) for HILIC-HPLC analysis.

[0132] 2.1.5 Preparation of reference solution

[0133] Take about 10 mg of fructose, sucrose and glucose, accurately weigh them, transfer them to a 10 mL volumetric flask, and dilute to the mark with water.

[0134] 2.2 Optimization of chromatographic conditions

[0135] 2.2.1 Detector selection

[0136] Compared with differential detection (HPLC-RID), HPLC-ELSD has many advantages, such as stable baseline, good separation effect, high response value, accurate results, and good reproducibility. In addition, the pretreatment for polysaccharide detection is relatively simple, and oligosaccharides can be separated and detected without pre-column derivatization. The results show that all peaks are separated, the peak shape is good, and the resolution is high.

[0137] 2.2.2 Column selection

[0138] In this study, a series of HILIC columns (Sunniest HILIC-S, Welch UltimateHILIC Amide and Hypersil GOLD TM We found that Hypersil GOLD TM PEI HILIC HPLC is suitable for the separation of Polygonatum sibiricum polysaccharide hydrolysate, with good separation resolution and peak distribution. Figure 11-13 .

[0139] 2.2.3 Investigation of column temperature

[0140] The study investigated the separation and peak shape of the same sample when the column temperature was set at 30℃, 35℃ and 40℃. The results showed that Figure 14-16The temperature has little effect on the separation and peak shape of the chromatographic peaks. When the column temperature is set at 35°C, the separation is slightly better than that at 30°C and 40°C. Therefore, the experimental column temperature is selected to be 35°C.

[0141] 2.2.4 Investigation of mobile phase and elution gradient

[0142] This study investigated the HPLC chromatographic peak separation effects of different samples under acetonitrile-water and methanol-water mobile phases. The results showed that the acetonitrile-water system had the best separation effect on Polygonatum sibiricum from different origins. In order to achieve the separation of the main chromatographic peaks of Polygonatum sibiricum from different origins, the elution gradient was further optimized in the experiment, and the linear elution mode was finally determined, as shown in Table 2. Figure 17-18 .

[0143] Table 2 Mobile phase gradient elution system

[0144]

[0145] 2.2.5 Investigation of the temperature of the evaporative light drift tube

[0146] This study investigated different drift tube temperatures of 90℃, 100℃, and 105℃. The experimental results showed that different drift tube temperatures have a significant impact on the baseline. Figures 19-21 When the drift tube temperature is 100℃, the baseline is relatively stable. The drift tube temperature is selected as 100℃ in the experiment.

[0147] 2.2.6 Investigation of injection volume

[0148] The study investigated the chromatographic separation effect and peak information quantity of the same batch of samples with different injection volumes of 5μL, 10μL, and 15μL. Figure 22-24 Comparison revealed that when the injection volume was 5 μL, the chromatographic peak area was small, and the chromatographic peaks with longer retention times were easily overlooked, which was not conducive to the subsequent analysis of oligosaccharide fragments. When the injection volume was 15 μL, the chromatographic peaks of some samples were poor. Considering all factors, the injection volume for this study was 10 μL.

[0149] 2.2.7 Determination of chromatographic conditions

[0150] Chromatographic column: Hypersil GOLD TM PEI HILIC HPLC (5μm, 250mm×4.6mm)

[0151] Mobile phase: acetonitrile (A), water (B);

[0152] Elution procedure: 0-25 min, 85%-65% A; 25-35 min, 65%-50% A;

[0153] Detector: Evaporative light detector

[0154] Flow rate: 1.0 mL min -1 ;

[0155] Column temperature: 35°C; injection volume: 10 μL

[0156] Under the above conditions, different sources of Polygonatum were well separated from other impurity peaks, and the separation degree from adjacent peaks was greater than 1.5. The reference and sample spectra are shown in Figures 25-28 .

[0157] 2.3 Methodological Investigation

[0158] 2.3.1 Precision test

[0159] Take one portion of the S2 test sample and prepare the test sample solution according to the content under "2.1". Inject the sample 6 times continuously and record the relative retention time and relative peak area of ​​each common peak (the peak time and peak area of ​​fructose are used as the reference, and the ratio of the peak time and peak area of ​​other peaks to it, the same below). The RSDs of the relative retention time and relative peak area of ​​the common peaks are both less than 3%, indicating that the instrument has good precision and meets the requirements of the fingerprint spectrum.

[0160] 2.3.2 Repeatability Experiment

[0161] Take 6 portions of S2 test sample, prepare the test sample solution according to the method under "2.1", and measure according to the chromatographic conditions under "2.1". The RSDs of the relative retention time and relative peak area of ​​each common peak are less than 3%. The results of each extraction of the same sample are relatively consistent, indicating that the extraction method has good reproducibility and meets the requirements of the fingerprint spectrum.

[0162] 2.3.3 Stability test

[0163] The same S2 test solution was taken and sampled at 0, 2, 4, 8, 12, and 24 hours respectively. The RSDs of the relative retention time and relative peak area of ​​the common peak were all less than 3%, indicating that the test solution was relatively stable within 24 hours, and the chemical composition and content would not change, which met the fingerprint requirements.

[0164] 3. Chemometric Analysis

[0165] 3.1.1 Hierarchical cluster analysis

[0166] The common peak areas of the HILIC-HPLC-ELSD fingerprints of 20 batches of Polygonatum sibiricum polysaccharides included in the pharmacopoeia were integrated to form a 20 × 17 data matrix. The data matrix was subjected to low-level fusion and imported into SPSS 23. Average correlation and Euclidean distance were used as clustering methods and similarity measures. Samples were classified based on individual differences.

[0167] 3.1.2 Principal Component Analysis

[0168] The 20 × 17 data matrix was imported into SPSS 23, and dimensionality reduction was used to obtain eigenvalues ​​and cumulative variance contributions. Variables with eigenvalues ​​> 1 were further selected for principal component analysis. The 20 × 17 data matrix was then imported into SIMCA 13 statistical software for PCA analysis.

[0169] 3.1.3 Partial Least Squares Discriminant Analysis

[0170] The 20 × 17 data matrix was imported into SIMCA 13 statistical software for PLS-DA analysis. The influence of each chromatographic peak on the identification results was determined by the VIP (Variable Importance in Projection) value.

[0171] 4 Results

[0172] In this study, 34 batches of samples were collected, including 5 batches of Polygonatum sibiricum, 5 batches of Polygonatum sibiricum from Yunnan, 10 batches of Polygonatum sibiricum from many flowers, 5 batches of Polygonatum sibiricum from Sichuan, 5 batches of Polygonatum sibiricum from Hubei, 2 batches of Polygonatum sibiricum from small leaves, and 2 batches of imported Polygonatum sibiricum, for HPLC-ELSD analysis. The differences between the three different types of Polygonatum sibiricum in the pharmacopoeia and whether they are different from the easily confused products in the market were identified.

[0173] 4.1 Fingerprint similarity evaluation

[0174] like Figures 29-31 As shown in Figure 2, HPLC-HILIC-ELSD detected 17 peaks corresponding to enzymatic hydrolysis products. By comparing the retention times of fructose, glucose and sucrose under the same conditions, the peaks were determined to be 17. Figure 7 , preliminarily determined that these peaks are DPs 1 to 7. The chromatograms of 5 batches of Polygonatum sibiricum, 5 batches of Polygonatum sibiricum, 10 batches of Polygonatum sibiricum, 5 batches of Polygonatum sibiricum from Sichuan, 5 batches of Polygonatum sibiricum from Hubei, 2 batches of Polygonatum sibiricum with small leaves, and 2 batches of imported Polygonatum sibiricum were imported into the Chinese Medicine Fingerprint Similarity Evaluation System (Version 2.0) software to generate reference spectra of the three original Polygonatum sibiricum. 34 batches of samples were compared with the reference spectra of Polygonatum sibiricum, Polygonatum sibiricum from Yunnan, and Polygonatum sibiricum, and the similarity was calculated. The results are shown in Tables 3-5. As can be seen from the figure:

[0175] ① Compared with the reference map of Polygonatum multiflorum, the similarity of 10 batches of Polygonatum multiflorum among the three original Polygonatum sibiricum is greater than 0.93. Compared with the reference map of Polygonatum multiflorum and Polygonatum acanthopanax, the similarity of Polygonatum yunnanensis and Polygonatum acanthopanax is less than 0.85, except for one batch of Polygonatum yunnanensis (D1). Compared with the reference map of Polygonatum multiflorum, the distribution of easily confused products on the market is more discrete, among which the similarity of Hubei Polygonatum is between 0.44 and 0.95; the similarity of Sichuan Polygonatum is less than 0.86 except for one batch (C4); the similarity of imported Polygonatum is less than 0.82; the similarity of Polygonatum microphylla is less than 0.5;

[0176] ②Compared with the reference spectrum of Polygonatum sibiricum, the similarity of the three ancestral Polygonatum species to the five batches of Polygonatum sibiricum was greater than 0.90. The similarity of Polygonatum sibiricum and Polygonatum multiflorum to the reference spectrum of Polygonatum sibiricum was less than 0.90, except for Polygonatum multiflorum (S1, S8, S9, S10) and Polygonatum sibiricum (J2). The similarity of other commercially available Polygonatum species to the reference spectrum ranged from 0.65 to 0.99, with the similarity of Sichuan Polygonatum all greater than 0.90; the similarity of Hubei Polygonatum and imported Polygonatum partially greater than 0.90; and the similarity of Polygonatum microphylla all less than 0.80.

[0177] ③Compared with the reference spectrum of Polygonatum cyrtonema, the similarity of the three ancestral Polygonatum species to the five batches of Polygonatum cyrtonema was >0.99. The similarity of Polygonatum cyrtonema and Polygonatum multiflorum to the three ancestral Polygonatum species, with the exception of Polygonatum multiflorum (S7) and Polygonatum cyrtonema (D4), was <0.80. The similarity of other commercially available Polygonatum species to the three ancestral Polygonatum species ranged from 0.60 to 0.99, with the similarity of Polygonatum cyrtonema from Sichuan being <0.80, and the similarity of Polygonatum cyrtonema from Hubei being <0.70, with the exception of H5. The similarity of Polygonatum cyrtonema from Hubei being >0.70, with the exception of imported Polygonatum cyrtonema and Polygonatum microphyllum being >0.90, with the exception of imported Polygonatum cyrtonema (Z2).

[0178] These results indicate that the fingerprint chromatograms within the three different sources of Polygonatum sibiricum listed in the Pharmacopoeia are highly similar, with minimal differences. The three original Polygonatum sibiricum species exhibit significant interspecific differences, while some easily confused products on the market also exhibit slight differences from the three Polygonatum sibiricum species listed in the Pharmacopoeia. Therefore, the structural characteristics of different Polygonatum sibiricum polysaccharides are distinct, making them easier to distinguish.

[0179] Table 3 Similarity evaluation results

[0180]

[0181] Note: The reference chromatogram of the Polygonatum multiflorum reference spectrum (R1) was used as a reference chromatogram, and the median method was used for multi-point calibration to generate the reference spectrum. The similarity of each batch was compared with R1. (S1-S10 Polygonatum multiflorum; J1-J5 Polygonatum acanthopanax; D1-D5 Polygonatum yunnanensis; C1-C5 Polygonatum sichuanensis; H1-H5 Polygonatum hupehensis; Z1-Z2 Polygonatum microphylla; X1-X2 Imported Polygonatum)

[0182] Table 4 Similarity evaluation results

[0183]

[0184]

[0185] Note: The reference chromatogram of the Polygonatum duniana reference spectrum (R2) was used as the reference spectrum. The median method was used for multi-point calibration to generate the reference spectrum. The similarity of each batch was compared with R2.

[0186] Table 5 Similarity evaluation results

[0187]

[0188] Note: The reference chromatogram of the Polygonatum duniana reference spectrum (R3) was used as the reference spectrum. The median method was used for multi-point calibration to generate the reference spectrum. The similarity of each batch was compared with R3.

[0189] 4.2 Cluster Analysis

[0190] The peak areas of the common peaks in the chromatograms of the enzymatic hydrolysis products of three Polygonatum varieties (a total of 20 batches) included in the pharmacopoeia were sorted to obtain a 20×17 order data matrix, which was imported into IBM SPSS Statistic version 23 software and clustered using inter-group linkage. The clustering results are shown in Figure 2. Figure 32 As shown, 20 batches of Polygonatum sibiricum samples were divided into three major categories: Category 1 included 10 batches of Polygonatum cyrtonema (Polygonatum multiflorum), sourced from Jinzhai and Shitai (3 samples) in Anhui Province, Hangzhou (2 samples), Linqi (2 samples), and Quzhou (2 samples) in Zhejiang Province, and Ruichang (2 samples) in Jiangxi Province; Category 2 included 5 batches of Polygonatum yunnanensis (Polygonatum yunnanensis), sourced from Dazhou and Chengdu (2 samples) in Sichuan Province, Wuhan in Hubei Province, Kunming in Yunnan Province, and Zhijin in Guizhou Province; and Category 3 included 5 batches of Polygonatum cyrtonema (Polygonatum acutiloba) ...

[0191] 4.3 Principal Component Analysis

[0192] The principal component analysis, which reflects the idea of ​​dimensionality reduction, selects components with larger contribution rates for analysis, which not only simplifies the analysis process but also displays as much original information as possible. The 17 common peak areas of 20 batches of samples were imported into SPSS 23.0 software for PCA analysis. The eigenvalues ​​and variance contribution rates of the principal component analysis are shown in Table 4, and the scree plot is shown in Table 4. Figure 33 . With the eigenvalue > 1 as the extraction standard, four principal components were extracted. The first three PCs accounted for 50.1%, 26.6% and 9.1% of the total variance respectively. Therefore, three principal components were selected for evaluation, which represented 85.8% of the information content of the 17 components in Polygonatum sibiricum polysaccharide, which was sufficient to evaluate the quality of Polygonatum sibiricum polysaccharide. The data matrix was imported into SIMCA statistical software, and three principal components were extracted. In this model, R 2 X(cum)=0.844,Q 2 (cum)=0.612, all greater than 0.5, indicating that the prediction ability of the PCA model is better. Figure 34) showed that the 20 batches of PCPs were classified into three categories, consistent with the HCA results. Polygonatum sibiricum S1-S10 clustered into one group. Polygonatum yunnanensis S11-S15 clustered into another group, while Polygonatum acicularis S16-S20 were assigned to a third group. The PCA model also provided satisfactory classification for the three different origins of Polygonatum sibiricum, demonstrating that enzymatic hydrolysis of Polygonatum sibiricum polysaccharides can effectively distinguish Polygonatum sibiricum from different sources.

[0193] Table 4 Eigenvalues ​​and variance contribution rates Total variance explained

[0194]

[0195]

[0196] 2.5.3 Partial Least Squares Discriminant Analysis

[0197] PLS-DA is often used for discriminant analysis of multivariate system samples and is suitable for situations where there is collinearity between independent variables or the number of samples is small. The data matrix is ​​imported into SIMCA statistical software for analysis through PLS-DA. In this model, R 2 X(cum)=0.909,R 2 Y(cum)=0.863,Q 2 (cum) = 0.775, R 2 Y and Q 2 The value of is preferably greater than 0.5, and R 2 Y and Q 2 The closer the value of is to 1, the better the prediction ability of the PLS-DA model is. Figure 35 As shown, the classification results are consistent with those of HCA and PCA. Figure 36 This reflects the model's VIP value. The VIP value indicates the contribution of each independent variable. Using a VIP value greater than 1 as the screening criterion, we identify the signature components that contribute to differences between production areas. The VIP values ​​for peaks 1, 4, 5, 7, and 8 are all greater than 1. These results indicate that these five peaks significantly contribute to the discriminant analysis of the PLS-DA model.

[0198] 5. Conclusion

[0199] Research has revealed confusion regarding the origins of Polygonatum sibiricum in the market. Among the three types of Polygonatum sibiricum listed in the Pharmacopoeia, Polygonatum multiflorum and Polygonatum acanthus present a degree of difficulty in distinguishing. Furthermore, there are cultivated variants of Polygonatum sibiricum and imported Polygonatum sibiricum. These variants differ in appearance from traditional Polygonatum sibiricum, yet conform to the characteristics described in the Pharmacopoeia, making them difficult to distinguish. Imported Polygonatum sibiricum, which primarily enters the Chinese market from countries like Myanmar and Vietnam, is larger and often marketed as Yunnan Polygonatum sibiricum, and contains a higher content. However, it remains unknown whether its efficacy and composition differ from the commonly used Polygonatum sibiricum in my country. Furthermore, a non-Pharmacopoeia variety called Evergreen Polygonatum has been widely cultivated in Guizhou, Sichuan, and other regions in recent years. Due to its rapid asexual reproduction, evergreen nature, ease of field management, and high yield, its cultivation is increasing. While this variety is not significantly different in appearance from the Pharmacopoeia variety, it is not a genuine Pharmacopoeia variety, and its efficacy and composition remain unknown. Therefore, this study conducted a comparative analysis of the Polygonatum sibiricum from different sources brought back for investigation, and established a method for the technical standard of the traditional Chinese medicine Polygonatum sibiricum based on the HPLC-HILIC-ELSD analysis of the enzymatic hydrolysis products of Polygonatum sibiricum polysaccharides. The fingerprint spectrum established in this study is related to the chemical composition and structural characteristics of Polygonatum sibiricum polysaccharides, and therefore has great practical value in the quality control of Polygonatum sibiricum polysaccharide samples.

[0200] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for establishing a multivariate fingerprint of Polygonatum sibiricum origin species, characterized in that: The method comprises the following steps: (1) Preparation of reference solution: Take appropriate amount of fructose, sucrose, and glucose reference substances, accurately weigh them, transfer them to a volumetric flask, and add water to the mark. (2) Preparation of test solution: Take an appropriate amount of Polygonatum sibiricum polysaccharide and mix it with fructosidase solution; hydrolyze the mixture in an oscillator, heat it, centrifuge it, take the supernatant, dry it, and dissolve it in acetonitrile-water solution to obtain the product; (3) Establishment of fingerprint: Use high performance liquid chromatography to detect the reference solution and the test solution, record the chromatogram, and establish the fingerprint of the reference solution and the test solution; the chromatographic conditions are as follows: Chromatographic column: Hypersil GOLD™ PEI HILIC HPLC, model: 5 μm, 250 mm × 4.6 mm; mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution; column temperature: 35 °C; flow rate: 1.0 mL min -1 ; Detector: Evaporative light scattering detector; injection volume 10 μL; Gradient elution conditions: 0-25 min, 85%-65% A; 25-35 min, 65%-50% A.

2. The method according to claim 1, characterized in that Preparation of the reference solution in step (1): Take 10 mg each of fructose, sucrose, and glucose reference substances, accurately weigh them, transfer them to a 10 mL volumetric flask, and dilute to the mark with water.

3. The method according to claim 1, characterized in that Preparation of the test solution in step (2): Take Polygonatum sibiricum polysaccharide, add water to make a Polygonatum sibiricum polysaccharide solution with a concentration of 0.5 mg / mL, add an equal volume of fructosidase solution and mix, place in an oscillator for hydrolysis, heat, centrifuge, take the supernatant, dry, and dissolve in 500 μL of acetonitrile-water solution to obtain.

4. The method according to any one of claims 1 or 3, characterized in that The concentration of the fructosidase solution in step (2) is 50-200 U / mL.

5. The method according to claim 4, characterized in that The concentration of the fructosidase solution in step (2) is 100 U / mL.

6. The method according to any one of claims 1 or 3, characterized in that The heating in step (2) is: heating at 80°C for 20 minutes.

7. The method according to any one of claims 1 or 3, characterized in that The setting conditions of the oscillator in step (2) are: temperature of 55°C, rotation speed of 200 rpm, and time of 1-7 hours.

8. The method according to claim 7, characterized in that The oscillator setting conditions in step (2) are: temperature of 55°C, rotation speed of 200 rpm, and time of 3 hours.

9. The method according to any one of claims 1 or 3, characterized in that The centrifugal setting conditions in step (2) are: 4500 r / min, 15 min.

10. The method according to any one of claims 1 or 3, characterized in that The volume ratio of acetonitrile to water in the acetonitrile-water solution in step (2) is 1:1.

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