Method for separating and detecting combined fructose in rhizoma polygonati

Abstract

The invention belongs to the technical field of analysis and detection of traditional Chinese medicinal materials, and particularly relates to a method for separating and detecting conjugated fructose in rhizoma polygonati. According to the method, interference substances are removed through pretreatment, chromatographic detection conditions are optimized, the content of the combined fructose in the rhizoma polygonati and the processed product of the rhizoma polygonati can be accurately measured, and the method is particularly suitable for evaluating the influence of processing technologies such as nine-steaming and nine-drying on the content of effective components of the rhizoma polygonati. Compared with the prior art, the method has the advantages of high sensitivity, good accuracy, strong repeatability and simplicity and convenience in operation, can be used for quality control and processing technology optimization of traditional Chinese medicinal materials (such as rhizoma polygonati), and provides a reliable detection means for standardization of traditional Chinese medicines.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis and detection technology, specifically relating to a method for separating and detecting bound fructose in Polygonatum sibiricum. Background Technology

[0002] Polygonatum, the dried rhizome of *Polygonatum kingianum* Coll. et Hemsl., *Polygonatum sibiricum* Red., or *Polygonatum cyrtonema* Hua, is an important traditional Chinese medicine. It is widely used in TCM clinical practice for its effects of tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. Modern pharmaceutical research shows that the medicinal activity of Polygonatum is closely related to its various chemical components. Among them, Polygonatum polysaccharides and Polygonatum flavonoids are the main marker active ingredients, while conjugated fructose, as a key component of saccharide active ingredients, plays a central role in the tonic and immunomodulatory effects of Polygonatum. Its content level directly relates to the quality and clinical efficacy of Polygonatum medicinal materials.

[0003] Currently, a certain technical system has been established for the detection of fructose. However, in the area of ​​accurate detection of bound fructose in Polygonatum sibiricum and its processed products, existing technologies still have significant shortcomings. Traditional detection methods generally suffer from insufficient sensitivity, making it difficult to accurately quantify low levels of bound fructose in complex matrices. Furthermore, various interfering substances in Polygonatum sibiricum extract, such as flavonoids and tannins, can severely interfere with the detection results, leading to poor data accuracy and repeatability. In addition, the cumbersome detection procedures and the weak specificity of pretreatment methods further limit detection efficiency. Crucially, during the traditional processing of Polygonatum sibiricum, such as the "nine steaming and nine drying" process, the content and structure of bound fructose undergo dynamic changes. Existing methods cannot adapt to the complex fluctuations in components during processing, making it difficult to meet the technical requirements for optimizing processing techniques and evaluating quality consistency.

[0004] With the advancement of modernization and standardization in the development of traditional Chinese medicinal materials, higher demands are being placed on testing technologies for the quality control and efficacy evaluation of Polygonatum sibiricum. The deficiencies of existing testing methods have become a key bottleneck restricting the standardized cultivation, optimization of processing techniques, and rational clinical application of Polygonatum sibiricum. Therefore, there is an urgent need to establish a highly sensitive, accurate, reproducible, and easy-to-operate method for detecting bound fructose. This method should effectively remove interfering substances from the Polygonatum sibiricum matrix, accurately measure the changes in bound fructose content at different processing stages, provide reliable technical support for the quality control and processing optimization of Polygonatum sibiricum, and ultimately promote the improvement of the standardization system for the quality of traditional Chinese medicine, meeting the core needs of modern production and clinical application of traditional Chinese medicinal materials. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a simple, accurate, and sensitive method for determining the content of bound fructose in Polygonatum sibiricum, so as to solve the problems of low sensitivity, large interference, and cumbersome procedures in the existing technology for detecting bound fructose.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: In a first aspect, the present invention provides a method for separating bound fructose from Polygonatum sibiricum, the method comprising: (1) Acidify and hydrolyze the extract of Polygonatum polysaccharide, and remove the acid to obtain the test solution; (2) The test solution was separated by high performance liquid chromatography to separate the bound fructose in Polygonatum odoratum; In step (2), the conditions for high performance liquid chromatography include: the mobile phase is acetonitrile-water, and the volume ratio of acetonitrile to water is (80:20)-(90:10).

[0007] Preferably, in the above method, the conditions for high-performance liquid chromatography further include one or more of the following conditions: (i) The chromatographic column is selected from Alltech Prevail Carbohydrate ES column, Hypersil GOLD™ PEIHILIC column or Waters XBridge® HILIC column, with column specifications of 4.6 × 250 mm and 5 μm; (ii) The injection volume is 5 μL-15 μL; (iii) The flow rate is 2.3 mL / min - 2.7 mL / min; (iv) Column temperature is 33℃-37℃.

[0008] More preferably, in the above method, the conditions for high performance liquid chromatography include: the mobile phase is acetonitrile-water, with a volume ratio of acetonitrile to water of 85:15; the chromatographic column is a WatersXBridge® HILIC column with dimensions of 4.6×250mm and 5μm; the flow rate is 2.5mL / min; and the column temperature is 35℃.

[0009] Preferably, in the above method, the acidification and hydrolysis conditions include: adding trifluoroacetic acid to the extract of Polygonatum polysaccharides and hydrolyzing at 105℃-115℃.

[0010] More preferably, in the above method, the acidification hydrolysis conditions further include one or more of the following conditions: (a) The volume ratio of Polygonatum polysaccharide extract to trifluoroacetic acid was 1:(1.5-2.5), and the concentration of trifluoroacetic acid was 0.01mol / L-1mol / L; (b) The acidification and hydrolysis time is 1h-3h.

[0011] More preferably, in the above method, the acidification hydrolysis conditions include: taking 1 mL of Polygonatum polysaccharide extract, adding 2 mL of 0.05 mol / L trifluoroacetic acid, and hydrolyzing at 110°C for 2 hours.

[0012] Preferably, the preparation method of the Polygonatum polysaccharide extract in the above method includes: adding Polygonatum powder to an ethanol solution, then refluxing and extracting in a water bath, cooling, centrifuging, and discarding the supernatant; washing the residue with an ethanol solution, centrifuging and discarding the supernatant, and retaining the residue; taking the residue, adding pure water, and heating and refluxing to extract the Polygonatum polysaccharide extract.

[0013] Secondly, this invention provides a method for detecting bound fructose in Polygonatum sibiricum, the method comprising: (1) Separate the Polygonatum polysaccharide extract according to the above method; (2) After separation, the sample enters the detector for detection; The detector is either an evaporative light scattering detector (ELSD) or a differential refractive index detector (RID).

[0014] Preferably, in the above method, the detector is an evaporative light scattering detector (ELSD), and the drift tube temperature of the evaporative light scattering detector is 95℃-120℃.

[0015] More preferably, in the above method, the detector is an evaporative light scattering detector (ELSD), and the drift tube temperature of the evaporative light scattering detector is 110°C.

[0016] The beneficial effects of this invention include at least the following: Compared with existing technologies, the method for separating and detecting bound fructose in Polygonatum sibiricum provided by this invention has the advantages of high sensitivity, good accuracy, strong repeatability, and simple operation. By removing interfering substances through pretreatment and optimizing chromatographic detection conditions, the content of bound fructose in Polygonatum sibiricum and its processed products can be accurately determined, and it is particularly suitable for evaluating the impact of processing techniques such as "nine steaming and nine sun-drying" on the content of effective components in Polygonatum sibiricum. The method of this invention can be used for the quality control and processing optimization of Chinese medicinal materials (such as Polygonatum sibiricum), providing a reliable detection method for the standardization of Chinese medicine. Attached Figure Description

[0017] Figure 1 This is a standard curve diagram for fructose reference standard; Figure 2 The chromatogram of the Polygonatum sibiricum test solution; Figure 3 The chromatogram is of the fructose reference solution. Figure 4 The chromatogram is the result of acid hydrolysis of 0.5 mL of Polygonatum polysaccharide extract. Figure 5 The chromatogram is the result of acid hydrolysis of 1 mL of Polygonatum polysaccharide extract; Figure 6 The chromatogram is the result of acid hydrolysis of 2 mL of Polygonatum polysaccharide extract; Figure 7 The chromatogram is obtained by hydrolyzing the polysaccharide extract with 1 mL of 0.01 mol / L trifluoroacetic acid solution. Figure 8 The chromatogram is obtained by hydrolyzing the polysaccharide extract with 2 mL of 0.01 mol / L trifluoroacetic acid solution. Figure 9 The chromatogram is obtained by hydrolyzing the polysaccharide extract with 3 mL of 0.01 mol / L trifluoroacetic acid solution. Figure 10 The chromatogram is obtained by hydrolyzing the polysaccharide extract with 1 mL of 0.05 mol / L trifluoroacetic acid solution. Figure 11 The chromatogram is obtained by hydrolyzing the polysaccharide extract with 2 mL of 0.05 mol / L trifluoroacetic acid solution. Figure 12 The chromatogram is obtained by hydrolyzing the polysaccharide extract with 3 mL of 0.05 mol / L trifluoroacetic acid solution. Figure 13 The chromatogram is obtained by hydrolyzing the polysaccharide extract with 1 mL of 0.1 mol / L trifluoroacetic acid solution. Figure 14 The chromatogram is obtained by hydrolyzing the polysaccharide extract with 2 mL of 0.1 mol / L trifluoroacetic acid solution. Figure 15 The chromatogram is obtained by hydrolyzing the polysaccharide extract with 3 mL of 0.1 mol / L trifluoroacetic acid solution. Figure 16 The chromatogram is obtained under the condition of hydrolysis time of 1 hour; Figure 17 The chromatogram was obtained under a hydrolysis time of 2 hours. Figure 18 The chromatogram is obtained under the condition of hydrolysis time of 3h; Figure 19 The chromatogram shows the separation of the hydrolysis products of Polygonatum odoratum using an Alltech Prevail Carbohydrate ES column; Figure 20 The chromatogram shows the separation of the hydrolysis products of Polygonatum odoratum using a Hypersil GOLD PEI HILIC column; Figure 21 The chromatogram shows the separation of the hydrolysis products of Polygonatum odoratum using a Waters XBridge® HILIC column; Figure 22 The chromatogram shows the chromatographic separation effect under the condition that the mobile phase acetonitrile-water volume ratio is 90:10. Figure 23The chromatogram shows the chromatographic separation effect under the condition that the mobile phase acetonitrile-water volume ratio is 85:15. Figure 24 The chromatogram shows the chromatographic separation effect under the condition that the mobile phase acetonitrile-water volume ratio is 80:20. Figure 25 The chromatogram is obtained under the condition that the drift tube temperature of the evaporative light detector is set to 95℃; Figure 26 The chromatogram is obtained under the condition that the drift tube temperature of the evaporative light detector is set to 110℃. Figure 27 The chromatogram is obtained under the condition that the drift tube temperature of the evaporative light detector is set to 120℃; Figure 28 Chromatograms obtained under injection volumes of 5 μL, 10 μL, and 15 μL; Figure 29 Chromatograms obtained under injection volumes of 5 μL, 10 μL, and 15 μL; Figure 30 The chromatograms are obtained under injection volumes of 5 μL, 10 μL, and 15 μL. Detailed Implementation

[0018] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0019] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0020] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0021] In the following example, the instrument information is as follows: U3000 high performance liquid chromatograph (equipped with an Agilent Alltech ELSD6000 evaporative light scattering detector), ML204 0.01% balance, XP26 0.01% balance (all instruments are provided by Mettler Toledo), Elmasonic S30H ultrasonic cleaner (Dexiang Technology Co., Ltd.), and Advantage A10 Milli-Q ultrapure water system (Merck Millipore).

[0022] In the following example, the reagent information is as follows: fructose reference standard (batch number 100231-202309, purity 99.8%, provided by the National Institutes for Food and Drug Control); acetonitrile (chromatographic grade), other reagents are analytical grade; water is laboratory ultrapure water.

[0023] Example 1 (1) Reagent preparation Raw Polygonatum medicinal material was processed using the traditional method of nine steaming and nine drying: cleaned fresh Polygonatum tubers were mixed with rice wine at 20% of the weight of Polygonatum and allowed to soak. Then, they were placed in a steamer and steamed until thoroughly cooked. After being slightly cooled, they were mixed with the distillate, cooled again, and then dried. This steaming and drying process was repeated 9 times. Finally, the processed Polygonatum was dried to constant weight, pulverized, and passed through a No. 3 sieve to obtain Polygonatum powder for later use. 2.0g of the above-processed Polygonatum powder was accurately weighed for content determination.

[0024] Preparation of the test solution for Polygonatum sibiricum: Add 2.0 g of the above Polygonatum sibiricum powder to 50 mL of 80% ethanol solution, reflux extract in an 80℃ water bath for 30 minutes, remove and cool to room temperature; centrifuge at 4500 rpm for 10 minutes, discard the supernatant; wash the residue twice with 80% ethanol solution, centrifuge and discard the supernatant, retain the residue; accurately weigh 1.0 g of the residue, add 50 mL of pure water, heat and reflux extract for 2 hours, combine the extracts after two extractions, and make up to 100 mL to obtain Polygonatum sibiricum polysaccharide extract; take 1 mL of the above Polygonatum sibiricum polysaccharide extract, add 2 mL of 0.05 mol / L trifluoroacetic acid, and hydrolyze at 110℃ for 2 hours; after hydrolysis, blow dry the residual liquid with nitrogen, then add anhydrous methanol to blow dry to remove excess acid, repeat the treatment 3 times; finally add an appropriate amount of water to make up to 2 mL, mix well and filter with a filter membrane to obtain the test solution for Polygonatum sibiricum.

[0025] Preparation of fructose reference stock solution: Accurately weigh 22.170 mg of fructose reference standard (batch number: 100231-202309, China National Institutes for Food and Drug Control), place it in a 25 mL volumetric flask, add pure water to dissolve and dilute to the mark, shake well to obtain the reference stock solution; as needed, dilute the reference stock solution appropriately to prepare fructose reference stock solutions of different concentration gradients for later use.

[0026] (2) Methodological verification The following methodological investigations were conducted on the fructose reference standard and the test sample. Chromatographic detection conditions: High performance liquid chromatography-evaporative light scattering (ELCS) was used for fructose content analysis. The chromatographic column was a WatersXBridge® HILIC column (4.6 × 250 mm, 5 μm), the mobile phase was acetonitrile-water (85:15 v / v), the column temperature was 35 °C, the mobile phase flow rate was 1.0 mL / min, the drift tube temperature of the evaporative light scattering detector was 110 °C, the carrier gas was air (flow rate 2.5 L / min), and the injection volume was 15 μL.

[0027] (2-1) Examination of linear relationships Different concentrations of fructose reference standard solutions (fructose reference standard stock solutions were serially diluted 2, 4, 8, and 16 times) were taken and injected under the chromatographic conditions described above to determine the peak area at each concentration. Linear regression analysis was performed with the logarithm of peak area as the ordinate (y) and the logarithm of reference standard concentration as the abscissa (x), and the fructose standard curve was obtained (see...). Figure 1 The regression equation is y = 1.9581x + 2.9993, and the correlation coefficient is... R 2 = 0.9994, indicating that the fructose concentration showed a good linear relationship with the peak area in the range of approximately 0.0554 mg / mL to 0.8868 mg / mL.

[0028] (2-2) Precision test Take 15 μL of the fructose reference solution prepared above and inject it 6 times consecutively, recording the chromatographic peak area. The relative standard deviation (RSD) of the fructose peak area was calculated to be 0.81%, indicating good instrument precision (see Table 1).

[0029] Table 1 Precision test of fructose reference standard

[0030] (2-3) Stability test The prepared Polygonatum sibiricum test solution (Y3) was injected at 0h, 2h, 4h, 6h, 8h, 10h and 12h after preparation to determine the fructose peak area. The RSD of the fructose peak area within 12 hours was calculated to be 1.1%, indicating that the test solution had good stability within 12 hours (see Table 2).

[0031] Table 2. Stability test of Polygonatum sibiricum sample

[0032] (2-4) Repeatability test Six parallel test solutions of Polygonatum sibiricum were prepared using the above method (Y3). The peak area of ​​fructose was measured and the content of bound fructose and RSD were calculated. This indicates that the method has good repeatability (see Table 3).

[0033] Table 3 Repeatability test of Polygonatum sibiricum sample

[0034] (2-5) Recovery rate test Weigh approximately 0.5 g of a sample of Polygonatum sibiricum (Y3) with known content, and accurately add 1 mL of a 21.34 mg / L fructose reference solution to prepare the test solution. Perform the determination in six parallel applications under chromatographic conditions to examine the method recovery rate of the analyte. The average recovery rate of fructose was 98.80%, with an RSD of 1.7%. This indicates that the method has good recovery (see Table 4).

[0035] Table 4. Fructose recovery rate test

[0036] (2-6) Content determination The method of this invention was used to determine the content of bound fructose in samples after different numbers of steaming and drying cycles (from 1 to 9 times) during the processing of Polygonatum sibiricum, and compared with unprocessed raw Polygonatum sibiricum samples. Typical chromatograms of the test samples are shown below. Figure 2 As shown, the chromatogram of the fructose reference solution is as follows. Figure 3 As shown in Table 5, the results of the determination of bound fructose content are listed in Table 5.

[0037] Table 5. Results of determination of bound fructose content in Polygonatum samples with different processing times (unit: mg / g)

[0038] As shown in Table 5, the content of bound fructose in Polygonatum sibiricum gradually decreased with the increase of processing and drying times. From the first to the third processing, the content of bound fructose decreased by about 35%, and then continued to decrease but tended to level off. Statistical analysis showed that the difference in bound fructose content among samples with different processing times was significant (for example, the difference between the first and third processed samples was significant). p <0.05; the difference between the 1st and 9th processed samples was extremely significant. p <0.01). The RSD of the test results for each group of samples was less than 3%, indicating that the method of the present invention has good repeatability and reliability for the content determination of Polygonatum sibiricum samples of different processing methods.

[0039] Example 2: Optimization Experiment of Detection Method To verify the reliability of the determination method of this invention, a systematic optimization experiment was conducted on the key conditions affecting the determination of bound fructose, including factors such as sample size, acid hydrolysis conditions, and chromatographic detection conditions. The specific implementation process is as follows. Optimization Experiment Method: Bound fructose needs to be released into free fructose through polysaccharide hydrolysis for detection; therefore, hydrolysis conditions and chromatographic conditions are the focus of method optimization.

[0040] (1) Hydrolysis conditions (1-1) Examination of sampling size 0.5 mL, 1 mL, and 2 mL of Polygonatum polysaccharide extract were respectively subjected to the above-mentioned acid hydrolysis treatment. Different Polygonatum test solutions were obtained according to the preparation method of the test solution of Polygonatum in Example 1. Then, the chromatographic detection conditions in Example 1 were used for detection to investigate the effect of different sampling volumes on the peak area of ​​bound fructose. The results are as follows. Figures 4 to 6 As shown in the figure. Considering both experimental signal intensity and ease of operation, 1 mL was selected as the optimal sampling volume for hydrolysis.

[0041] (1-2) Investigation of acid concentration and dosage One mL of Polygonatum sibiricum polysaccharide extract processed three times was used as a sample. 1 mL, 2 mL, and 3 mL of 0.01 mol / L, 0.05 mol / L, and 0.1 mol / L trifluoroacetic acid solutions were added respectively, and the mixture was hydrolyzed at 110℃ for 2 h. Different Polygonatum sibiricum test solutions were obtained according to the preparation method of the test solution in Example 1. Then, the chromatographic detection conditions in Example 1 were used for detection. The results showed that the peak area of ​​bound fructose was largest when 2 mL of 0.05 mol / L trifluoroacetic acid solution was added; excessively high acid concentration (0.1 mol / L) led to partial degradation of fructose, resulting in a decrease in peak area (see...). Figures 7 to 15 Therefore, the optimal conditions for acid hydrolysis were determined to be 2 mL of 0.05 mol / L trifluoroacetic acid for 2 hours.

[0042] (1-3) Investigation of hydrolysis time Under the optimal acid dosage (2 mL of 0.05 mol / L trifluoroacetic acid), different Polygonatum sibiricum test solutions were obtained according to the preparation method of the test solution in Example 1. The effects of hydrolysis for 1 h, 2 h, and 3 h on the release of bound fructose were investigated. That is, the Polygonatum sibiricum test solutions with different hydrolysis times were detected according to the chromatographic detection conditions in Example 1. The results are as follows: Figures 16 to 18 As shown in the figure. The results indicate that the peak area of ​​bound fructose reaches its maximum after 2 hours of hydrolysis, and extending the hydrolysis time to 3 hours does not significantly increase the peak area of ​​fructose. Therefore, the hydrolysis time was determined to be 2 hours.

[0043] (2) Investigation of chromatographic conditions The preparation method of the test solution of Polygonatum sibiricum is as follows: Following the method in Example 1, 1 mL of Polygonatum sibiricum polysaccharide extract was mixed with 2 mL of 0.05 mol / L trifluoroacetic acid and hydrolyzed in an oven at 110 °C for 2 h. After hydrolysis, the solution was dried under nitrogen and then dried under methanol to remove acid. This process was repeated three times. Water was then added to a final volume of 2 mL to obtain the test solution of Polygonatum sibiricum. After filtration through a filter membrane, the solution was used for chromatographic analysis.

[0044] The fructose reference standard stock solution was prepared as follows: Accurately weigh 22.170 mg of fructose reference standard, dissolve it in water, and dilute to 25 mL to obtain the fructose reference standard stock solution. This stock solution was used to establish a standard curve and for methodological investigation.

[0045] (2-1) Detector selection The effects of evaporative light scattering detector (ELSD) and refractive index detector (RID) on the detection of bound fructose in the test solution of Polygonatum sibiricum sample were compared, with other chromatographic conditions the same as in Example 1. The results showed that ELSD had a stable baseline, high sensitivity, and good reproducibility, and could be directly used for the detection of bound fructose without the need for cumbersome pre-column derivatization. Therefore, ELSD was selected as the detector in this method.

[0046] (2-2) Selection of chromatographic column The effectiveness of various chromatographic columns in separating bound fructose from the test solution of Polygonatum sibiricum was investigated, including an Alltech Prevail Carbohydrate ES column, a Hypersil GOLD™ PEI HILIC column, and a Waters XBridge® HILIC column (all 4.6 × 250 mm, 5 μm). Other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figures 19 to 21 As shown, the Waters XBridge® HILIC column provides the best resolution and peak shape for each sugar component, hence this column was selected.

[0047] (2-3) Optimization of mobile phase system Using acetonitrile-water as the mobile phase, the effect of different ratios (90:10, 85:15, and 80:20, v / v) on the separation of bound fructose in the test solution of Polygonatum sibiricum was investigated. Other chromatographic conditions were the same as in Example 1. The results are as follows: Figures 22 to 24 As shown in the figure. The results indicate that the peak shape and separation of each component are optimal under the acetonitrile-water (85:15) condition, therefore the mobile phase ratio of 85:15 was determined.

[0048] (2-4) Investigation of drift tube temperature The effect of drift tube temperatures (95℃, 110℃, and 120℃) on the baseline stability of bound fructose in the test solution of Polygonatum sibiricum was investigated. Other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figures 25 to 27As shown in the figure, the baseline noise was lowest and most stable at 110℃, so the drift tube temperature was selected as 110℃.

[0049] (2-5) Investigation of injection volume The effects of injection volumes of 5 μL, 10 μL, and 15 μL on the peak shape and detection signal of bound fructose in the test solution of Polygonatum sibiricum were investigated. Other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figures 28 to 30 As shown, the results showed that with an injection volume of 15 μL, the peak shapes of each component were good and the signals were strong. Increasing the injection volume further did not significantly improve the detection sensitivity. Taking all factors into consideration, the injection volume was determined to be 15 μL.

[0050] As can be seen from the above, the final preferred chromatographic conditions in this invention are as follows: the chromatographic column is a WatersXBridge® HILIC column (4.6×250mm, 5μm); the mobile phase is acetonitrile-water (85:15); the flow rate is 2.5mL / min; the column temperature is 35℃; the detector is an evaporative light scattering detector (drift tube temperature 110℃, carrier gas is air); and the injection volume is 15μL.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for separating bound fructose from Polygonatum sibiricum, characterized in that the method... include: (1) Acidify and hydrolyze the extract of Polygonatum polysaccharide, and remove the acid to obtain the test solution; (2) The test solution was separated by high performance liquid chromatography to separate the bound fructose in Polygonatum odoratum; In step (2), the conditions for high performance liquid chromatography include: the mobile phase is acetonitrile-water, and the volume ratio of acetonitrile to water is (80:20)-(90:10).

2. The method according to claim 1, characterized in that, High-performance liquid chromatography (HPLC) conditions also include one or more of the following: (i) The chromatographic column is selected from Alltech Prevail Carbohydrate ES column, Hypersil GOLD™ PEI HILIC column or Waters XBridge® HILIC column, with column specifications of 4.6 × 250 mm and 5 μm; (ii) The injection volume is 5 μL-15 μL; (iii) The flow rate is 2.3 mL / min - 2.7 mL / min; (iv) Column temperature is 33℃-37℃.

3. The method according to claim 2, characterized in that, The conditions for high performance liquid chromatography (HPLC) included: the mobile phase was acetonitrile-water with a volume ratio of 85:15; the chromatographic column was a WatersXBridge® HILIC column with dimensions of 4.6 × 250 mm and a diameter of 5 μm; the flow rate was 2.5 mL / min; and the column temperature was 35 °C.

4. The method according to any one of claims 1 to 3, characterized in that, The acidification and hydrolysis conditions include: adding trifluoroacetic acid to the extract of Polygonatum polysaccharides and hydrolyzing at 105℃-115℃.

5. The method according to claim 4, characterized in that, Acidification hydrolysis conditions also include one or more of the following: (a) The volume ratio of Polygonatum polysaccharide extract to trifluoroacetic acid was 1:(1.5-2.5), and the concentration of trifluoroacetic acid was 0.01mol / L-1mol / L; (b) The acidification and hydrolysis time is 1h-3h.

6. The method according to claim 5, characterized in that, The acidification and hydrolysis conditions included: taking 1 mL of Polygonatum polysaccharide extract, adding 2 mL of 0.05 mol / L trifluoroacetic acid, and hydrolyzing at 110℃ for 2 hours.

7. The method according to claim 1, 2, 3, 5 or 6, characterized in that, The preparation method of Polygonatum polysaccharide extract includes: adding Polygonatum powder to an ethanol solution, then refluxing and extracting in a water bath, cooling, centrifuging, and discarding the supernatant; washing the residue with an ethanol solution, centrifuging and discarding the supernatant, and retaining the residue; taking the residue, adding pure water, heating and refluxing to extract Polygonatum polysaccharide extract.

8. A method for detecting bound fructose in Polygonatum sibiricum, characterized in that the method... include: (1) Separate the Polygonatum polysaccharide extract according to the method described in any one of claims 1 to 7; (2) After separation, the sample enters the detector for detection; The detector is either an evaporative light scattering detector (ELSD) or a differential refractive index detector (RID).

9. The method according to claim 8, characterized in that, The detector is an evaporative light scattering detector (ELSD), and the drift tube temperature of the evaporative light scattering detector is 95℃-120℃.

10. The method according to claim 9, characterized in that, The detector is an evaporative light scattering detector (ELSD), and the drift tube temperature of the evaporative light scattering detector is 110℃.

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