Method for measuring content of inulin fructan in codonopsis pilosula

Abstract

The invention discloses a method for determining the content of inulin fructan in codonopsis pilosula. The method comprises the following four steps: (1) preparing total polysaccharide of codonopsis pilosula; (2) preparing a codonopsis pilosula and inulin type fructan reference substance; (3) establishing a linear curve equation of reference substance quality-chromatographic peak area by combining HPGPC with a differential detector, and determining the availability of the method through methodology investigation; and (4) determining the content of inulin fructan in codonopsis pilosula from different producing areas. The method is applied to quality evaluation of codonopsis pilosula medicinal materials and decoction pieces, and is applied to determination of the content of inulin fructan in a compound preparation containing codonopsis pilosula.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation and content determination of traditional Chinese medicine components, relates to Codonopsis pilosula, and specifically is a method for determining the content of inulin-type fructan in Codonopsis pilosula. Background Art

[0002] Codonopsis pilosula Codonopsis pilosula (Franch.) Nannf.、 Codonopsis pilosula C. pilosula Nannf. var. modesta (Nannf.) LTShen or Codonopsis pilosula C.tangshen The dried root of Oliv. is mild in nature and sweet in taste, and has the effects of tonifying the middle and replenishing qi, strengthening the spleen and lungs, promoting the production of body fluids and quenching thirst. The chemical components of Codonopsis pilosula mainly include polysaccharides, polyacetylenes, triterpenes, phenylpropanoids and alkaloids. Among them, Codonopsis pilosula polysaccharide is one of the main active ingredients of Codonopsis pilosula, which has multiple pharmacological effects such as immunomodulation, anti-oxidation, anti-aging, anti-inflammatory and regulation of intestinal flora.

[0003] The 2020 edition of the Chinese Pharmacopoeia stipulates the properties, identification, inspection and extracts of Codonopsis pilosula, but lacks content determination. Codonopsis pilosula polysaccharides are important active ingredients in Codonopsis pilosula, among which inulin-type fructans are the main components of Codonopsis pilosula polysaccharides, and their content is crucial to the quality of Codonopsis pilosula medicinal materials. At present, there are relatively many studies on the content determination of total polysaccharides in Codonopsis pilosula, and the main method used is the phenol-sulfuric acid method. However, this method is a colorimetric method, which has large errors, more interferences, and poor reproducibility in actual operation.

[0004] At present, there is no report on the determination method of inulin-type fructan content in Codonopsis pilosula. Summary of the invention

[0005] The invention aims to provide a method for determining the content of inulin-type fructan in Codonopsis pilosula, which is used for quality evaluation of Codonopsis pilosula medicinal materials and decoction pieces and quality evaluation of compound preparations containing Codonopsis pilosula.

[0006] The present invention is achieved by adopting the following technical solutions: A method for determining the content of inulin-type fructan in Codonopsis pilosula comprises the following steps: Step (1), preparation of total polysaccharides from Codonopsis pilosula The Codonopsis pilosula is extracted with a methanol-ethyl acetate extract, the residue is filtered out, the methanol-ethyl acetate extract is collected, and the methanol-ethyl acetate is recovered; The residue was extracted by reflux extraction with distilled water, concentrated to 1 / 4 to 1 / 3 of the volume by reduced pressure, and then precipitated with ethanol. The supernatant was removed by centrifugation, and the precipitate was washed with ether, ethyl acetate, acetone, and anhydrous ethanol in sequence, and finally freeze-dried to obtain a total polysaccharide sample of Codonopsis pilosula, which was weighed and the weight was recorded.

[0007] Further preferably, the volume ratio of methanol to ethyl acetate in the methanol-ethyl acetate extract is 1:0.2 to 1:0.5; the solid-liquid ratio of Codonopsis pilosula and the methanol-ethyl acetate extract is 1:5 to 1:8.

[0008] The solid-liquid ratio of the medicinal residue to distilled water is 1:5-1:10; the ethanol used is 60%-90% ethanol; and the centrifugal speed is 1000-1500 rpm.

[0009] Step (2), preparation of Codonopsis pilosula inulin-type fructan reference substance The total polysaccharide of Codonopsis pilosula is dissolved in ethanol, and then ultrafiltration is repeated for multiple times through a 5000Da molecular weight ultrafiltration membrane. The speed of the peristaltic pump is 10-20rpm. When the filtrate is detected by high performance gel permeation chromatography as a single chromatographic peak, it is determined that the inulin-type fructan reference substance is completely prepared. The ultrafiltrate is concentrated and freeze-dried to obtain the Codonopsis pilosula inulin-type fructan reference substance.

[0010] More preferably, 30% to 60% ethanol is used and dissolved to 50 mg / L to 100 mg / L.

[0011] Codonopsis pilosula inulin type fructan reference substance 1 H-NMR, 13 C-NMR and DEPT identified it as inulin-type fructan. 1 The degree of polymerization was determined by H-NMR.

[0012] Step (3), establishing a linear curve equation of the reference substance mass-chromatographic peak area by HPGPC combined with a differential detector; Accurately weigh 1.0 mg of Codonopsis pilosula inulin-type fructan reference substance and dissolve it in 1.0 mL of ultrapure water to obtain a 1 mg / mL reference substance solution; The reference solution was accurately aspirated and filtered using a 0.45 μm microporous filter membrane; 10.0 μL, 8.0 μL, 6.0 μL, 4.0 μL, and 2.0 μL were injected for HPGPC analysis, and the peak area and retention time were recorded. The standard curve was drawn with mass (X) and peak area (Y), and the linear regression equation Y = 6.27 × 10 7 X-3545.75, R 2 =0.99994.

[0013] HPGPC chromatographic conditions: The chromatographic conditions were Agilent 1260 HPLC system, chromatographic column: 7.8 mm × 30 cm, 10 μm; detector: differential refractive index detector; mobile phase: ultrapure water; flow rate 0.3 ml / min; column temperature: 35 °C; detector temperature: 35 °C.

[0014] Step (4), determining the inulin-type fructan content in Codonopsis pilosula from different origins For the Codonopsis pilosula samples to be tested, 3 portions are taken from each batch to prepare the test solution; then HPGPC analysis is performed (the HPGPC chromatographic conditions are the same as those in step (3)), and the peak area of ​​the chromatographic peak at the corresponding position of the reference substance is recorded and its content is calculated; The preparation method of the test solution is as follows: 1) The Codonopsis pilosula sample to be tested is extracted with methanol-ethyl acetate extract at a solid-liquid ratio of 1:5 to 1:8, and the drug residue is filtered to obtain the drug residue, and the methanol-ethyl acetate extract is collected and the methanol-ethyl acetate is recovered; wherein the ratio of methanol to ethyl acetate in the methanol-ethyl acetate extract is 1:0.2 to 1:0.5; 2) The drug residue is reflux extracted with distilled water at a solid-liquid ratio of 1:5 to 1:10, extracted 3 times, and the 3 extractions are combined, concentrated to 1 / 4 to 1 / 3 of the volume under reduced pressure, precipitated with 60% to 90% ethanol, centrifuged at 1000 to 1500 rpm for 15 to 30 minutes, the supernatant is removed, and the precipitate is washed 3 times with ether, ethyl acetate, acetone, and anhydrous ethanol in sequence, and freeze-dried to obtain a Codonopsis pilosula total polysaccharide sample, and weighed and the weight is recorded; 3) Accurately weigh the Codonopsis pilosula total polysaccharide sample, dissolve it in ultrapure water, and obtain the test solution.

[0015] The mechanism by which the method of the present invention can achieve the quantitative determination of inulin-type fructan in Codonopsis pilosula is that, first, the content of inulin-type fructan in Codonopsis pilosula is relatively high, and its absorption peak can be displayed in the total polysaccharide HPGPC chromatogram and the peak area integration can be accurately performed; secondly, the Codonopsis pilosula inulin-type fructan reference substance can be accurately prepared and obtained. At present, it is reported that there are many types of polysaccharides contained in Codonopsis pilosula, and other types of polysaccharides do not meet the requirements of the above two aspects, so they cannot be quantitatively determined. There are also reports on inulin-type fructan in Codonopsis pilosula in the existing literature, but they only involve chemistry, not quantification, and the inulin-type fructan in Codonopsis pilosula reported in the literature has different polymerization degrees due to the different preparation methods adopted, and it is impossible to achieve a one-to-one correspondence between the inulin-type fructan reference substance and the inulin-type fructan in the total polysaccharide. Therefore, the present invention first innovates three coherent processes of total polysaccharide preparation of Codonopsis pilosula-preparation of inulin-type fructan-standard curve formulation, and achieves accurate quantitative determination of inulin-type fructan in Codonopsis pilosula.

[0016] The invention is reasonably designed, and the method has good practical application value in the quality evaluation of Codonopsis pilosula medicinal materials and decoction pieces and the determination of the inulin-type fructan content in the compound preparation containing Codonopsis pilosula. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Indications for the reference substance of Codonopsis pilosula inulin-type fructan 1 H-NMR (A), 13 C-NMR (B) and DEPT135 (C).

[0020] in, Figure 1 A in the middle is the characteristic of inulin-type fructan 1 H-NMR, the signal at the chemical shift of 5.35ppm is the hydrogen signal of the glucose terminal group at the end of the inulin fructan structure; the signals in the chemical shift range of 3.0-4.5ppm are the hydrogen signals of the 1st, 3rd, 4th, 5th and 6th positions of the fructan inulin-type fructan.

[0021] Figure 1 B is the characteristic of inulin-type fructan 13 C-NMR, the chemical shifts from large to small are the signals of inulin-type fructan C-2 (103.20ppm), C-5 (81.04ppm), C-3 (76.95ppm), C-4 (74.25ppm), C-6 (62.09ppm), and C-1 (60.86ppm). The weak signal with a chemical shift of 92.45ppm is the glucose terminal carbon signal at the end of the inulin fructan structure.

[0022] Figure 1 The disappearance of the signal at the chemical shift of 103.20 and the inverted peaks at the chemical shifts of 62.09ppm and 60.86ppm in C and B indicate the presence of fructosyl. The integral ratio of the chemical shift of 4.18ppm signal peak to the chemical shift of 5.35 signal peak is 20:1, which indicates that the inulin-type fructan polymer is about 21.

[0023] Figure 2 The results of specificity investigation are shown as follows: HPGPC chromatograms of the reference substance (A) and the test substance (B).

[0024] in, Figure 2 A in the figure indicates that the inulin-type fructan HPGPC has a single chromatographic peak, indicating that the inulin-type fructan was successfully prepared.

[0025] Figure 2 Figure B shows that the absorption peak of inulin-type fructan in total polysaccharides of Codonopsis pilosula is not interfered by other substances and can be quantified.

[0026] Figure 3The results of the linear relationship investigation are shown; (A): HPGPC chromatogram of reference solution of different masses (10.0µg, 8.0µg, 6.0µg, 4.0µg, 2.0µg), (B): linear curve; the results show good linearity in the range of 2µg to 10µg.

[0027] Figure 4 HPGPC spectrum of total polysaccharides obtained by water extraction-alcohol precipitation method.

[0028] Figure 5 HPGPC chromatogram (A) and silica gel thin layer chromatogram (B) showing the shoulder peak. DETAILED DESCRIPTION

[0029] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0030] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0031] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0032] The embodiment of the present invention is to scientifically and rationally extract the total polysaccharides of Codonopsis pilosula and accurately prepare and identify the Codonopsis pilosula inulin-type fructan reference substance, then use high performance gel permeation chromatography (HPGPC) combined with a differential detector to establish a linear curve equation between its mass and chromatographic peak area, and determine the feasibility of the method through methodological investigation; the content of inulin-type fructan in 20 batches of Codonopsis pilosula medicinal materials is determined by this method, and the feasibility of the method is confirmed.

[0033] 1. First, the present invention aims to solve the following three technical problems (1) Preparation of Codonopsis pilosula polysaccharides If the HPGPC content of inulin-type fructan in total polysaccharides of Codonopsis pilosula is to be determined, the preparation method of total polysaccharides of Codonopsis pilosula is very critical. If the conventional water extraction-alcohol precipitation preparation method is used, a shoulder peak will appear after the inulin-type fructan chromatogram in the HPGPC spectrum of total polysaccharides (such as Figure 4 As shown), at this time, it is impossible to accurately quantify the absorption peak area of ​​inulin-type fructan, and thus it is impossible to establish a content determination method. Therefore, the embodiment of the present invention separates the shoulder peak at the back and compares it by silica gel thin layer chromatography, identifying the shoulder peak as fructose monosaccharide (such as Figure 5 as shown).

[0034] Therefore, in order to eliminate the impact of the shoulder peak, the embodiment of the present invention adds a step of methanol-ethyl acetate extraction before the water extraction of Codonopsis pilosula to remove monosaccharides such as fructose and glucose contained in Codonopsis pilosula without affecting polysaccharides, thereby achieving the goal that Codonopsis pilosula inulin-type fructans are free from interference from other chromatographic peaks in the HPGPC chromatogram of Codonopsis pilosula total polysaccharides, and the peak area of ​​the chromatographic peak can be accurately determined (such as Figure 2 -B).

[0035] (2) Two technical issues involved in the preparation of Codonopsis pilosula inulin-type fructan reference substances The first is what solvent to use to dissolve the total polysaccharide and then perform ultrafiltration. If water is used to dissolve the total polysaccharide, on the one hand, water as a solvent can dissolve the super-large molecular weight polysaccharide in the total polysaccharide. When performing ultrafiltration, the ultrafiltration membrane is easily blocked due to the presence of super-large molecules, affecting the ultrafiltration process; on the other hand, when water is used as a solvent, repeated ultrafiltration is required, which takes 3 to 5 days, and the aqueous solution is easy to deteriorate, affecting the experimental results. Therefore, the embodiment of the present invention selects to use a certain proportion of ethanol to dissolve the total polysaccharide, which not only solves the problem of ultrafiltration membrane blockage, but also solves the problem of solution deterioration.

[0036] The second is to choose the pore size of the ultrafiltration membrane. After screening 10000Da, 5000Da and 3000Da ultrafiltration membranes, the 5000Da ultrafiltration membrane was finally selected. Because the pore size of the 10000Da ultrafiltration membrane is too large, it is impossible to obtain the pure inulin-type fructan reference substance (with impurities); the pore size of the 3000Da ultrafiltration membrane is too small, the ultrafiltration is seriously blocked, and it is impossible to continue ultrafiltration.

[0037] (3) Create a linear curve After solving the above two technical problems, the embodiment of the present invention conducted a linear relationship between the mass and peak area of ​​Codonopsis pilosula inulin-type fructans. Through exploration, it was finally established that Codonopsis pilosula inulin-type fructans have good linearity in the range of 2.0µg to 10.0µg, which can be quantitatively determined, and the methodological investigation meets the requirements.

[0038] By solving the above three technical problems, the inulin-type fructan chromatogram peak in the HPGPC chromatogram of Codonopsis pilosula total polysaccharides is free of any interfering peaks, and its peak area can be accurately integrated; at the same time, the inulin-type fructan at this chromatographic peak is accurately prepared to obtain a qualified reference substance; the inulin-type fructan reference substance is proportional to the peak area (linear relationship) within a certain mass range, thereby achieving quantification.

[0039] 2. The method for determining the content of inulin-type fructan in Codonopsis pilosula is as follows: Step (1), extraction of total polysaccharides from Codonopsis pilosula The Codonopsis pilosula is extracted with methanol-ethyl acetate extract (the volume ratio of methanol to ethyl acetate is 1:0.2-1:0.5) at a solid-liquid ratio of 1:5-1:8, the residue is filtered out, the methanol-ethyl acetate extract is collected, and the methanol-ethyl acetate is recovered.

[0040] The residue was extracted with distilled water at a solid-liquid ratio of 1:5-1:10, and the extraction was performed 3 times. The 3 extractions were combined and concentrated to 1 / 4-1 / 3 volume under reduced pressure, precipitated with 60%-90% ethanol, centrifuged at 1000-1500 rpm for 15-30 min, the supernatant was removed, and the precipitate was washed 3 times with ether, ethyl acetate, acetone and anhydrous ethanol in sequence, and freeze-dried to obtain a total polysaccharide sample of Codonopsis pilosula, which was weighed and the weight was recorded.

[0041] Step (2), preparation of inulin-type fructan reference substance The total polysaccharide sample of Codonopsis pilosula was dissolved in 30% to 60% ethanol to a certain concentration (50 mg / L to 100 mg / L), and then ultrafiltered repeatedly through a 5000Da molecular weight ultrafiltration membrane at a peristaltic pump speed of 10 to 20 rpm. When the filtrate was detected by high performance gel permeation chromatography (HPGPC) as a single chromatographic peak, it was determined that the preparation was complete. The ultrafiltrate was concentrated and freeze-dried to obtain an inulin-type fructan reference substance.

[0042] NMR identification of Codonopsis pilosula inulin-type fructan reference substance: Codonopsis pilosula inulin-type fructan reference substance (50 mg) was dissolved in 1 mL of deuterated water (D2O) and analyzed by 400 MHz NMR spectrometer. 1 H-NMR, 13 C-NMR, 13 C-DEPT135 measurement, the NMR spectrum of the inulin-type fructan reference substance was obtained. 1 H-NMR, 13 C-NMR and DEPT135 identified it as inulin-type fructan with a degree of polymerization of about 21. Figure 1 shown.

[0043] Step (3): Establishment of linear curve of inulin-type fructan and methodological investigation 3.1 Preparation of reference solution Accurately weigh 1.0 mg of Codonopsis pilosula inulin-type fructan reference substance and dissolve it in 1.0 mL of ultrapure water to obtain a 1 mg / mL reference substance solution.

[0044] 3.2 Preparation of test solution Preparation of the test solution of total polysaccharide from Codonopsis pilosula produced in Shanxi: Accurately weigh 1.0 mg of Codonopsis pilosula polysaccharide powder (prepared by the method in step (1)) and dissolve it in 1.0 mL of ultrapure water to obtain a 1 mg / mL test solution.

[0045] Preparation of the test solution of total polysaccharide from Codonopsis pilosula produced in Gansu: Accurately weigh 2.5 mg of Codonopsis pilosula polysaccharide powder (prepared by the method in step (1)) and dissolve it in 1.0 mL of ultrapure water to obtain a 2.5 mg / mL test solution.

[0046] 3.3 Methodological validation (a) Specificity test Accurately pipette 10.0 μL of each reference solution and test solution, and perform HPGPC analysis under the following chromatographic conditions.

[0047] HPGPC chromatographic conditions: The chromatographic conditions were Agilent 1260 HPLC system, chromatographic column: TSK gel G4000PWXL (7.8mm×30cm, 10μm); detector: differential refractometer (1260 RID-G1362A); mobile phase: ultrapure water; flow rate 0.3ml / min; column temperature: 35℃; detector temperature: 35℃; injection volume 10.0μL.

[0048] The results showed that under this chromatographic condition, the separation of each component was good and the method specificity was high. Figure 2 shown.

[0049] (b) Linear relationship Accurately pipette the reference solution and filter it with a 0.45 μm microporous membrane. Take 10.0 μL, 8.0 μL, 6.0 μL, 4.0 μL, and 2.0 μL for injection respectively, perform HPGPC analysis according to the above chromatographic conditions, record the peak area and retention time, and draw a standard curve with mass (X) and peak area (Y). The linear regression equation Y=6.27×10 7 X-3545.75, R 2 =0.99994, indicating that the linear relationship of Codonopsis pilosula inulin-type fructan is good between 2μg and 10μg. Figure 3 shown.

[0050] (c) Precision test Weigh 1.25 mg, 0.64 mg, and 0.28 mg of reference substance accurately and dissolve them in 1 mL of ultrapure water to prepare reference substance solutions of different concentrations. Take 10 μL of each reference substance solution of different concentrations, inject 6 times continuously, perform HPGPC analysis according to the above chromatographic conditions, and record the peak area. The calculation shows that RSD < 3%, indicating that the intra-day precision of the instrument is good. The results are shown in Table 1.

[0051] Table 1 Precision investigation results

[0052] (d) Stability test Accurately pipette 10 μL of the Codonopsis pilosula test solution and the reference solution and store them at room temperature. Inject the samples at 0h, 3h, 6h, 12h, 24h, and 48h, perform HPGPC analysis according to the above chromatographic conditions, record the peak area, and calculate the RSD of the peak area. The results show that the RSD of the test solution and the reference solution are 2.8% and 2.2%, respectively. This shows that under room temperature conditions, the test and reference solutions have good stability within 48 hours, and the results are shown in Table 2.

[0053] Table 2 Stability test results

[0054] (e) Repeatability test Accurately weigh 1.32 mg, 0.72 mg, and 0.33 mg of total polysaccharides from Codonopsis pilosula and dissolve them in 1 mL of ultrapure water to prepare test solutions of different concentrations. Take 10 μL of each reference solution of different concentrations and inject it three times continuously. Perform HPGPC analysis according to the above chromatographic conditions and record the peak area of ​​the chromatographic peak at the corresponding position of the reference solution. The RSD of the peak area per unit mass of inulin-type fructans from Codonopsis pilosula was calculated to be <2%, indicating that the method has good repeatability. The results are shown in Table 3.

[0055] Table 3 Repeatability test results

[0056] (f) Sample recovery test Accurately weigh 9 portions of the same batch of Codonopsis polysaccharide powder, 0.6 mg per portion. Add the reference substance at 80%, 100%, and 120% of the known content to prepare the test solution, prepare 3 portions in parallel for each ratio, perform HPGPC analysis according to the above chromatographic conditions and record the chromatographic peak area of ​​the corresponding position of the reference substance. Calculate the average sample recovery rate of Codonopsis inulin-type fructan, and the results show that the method has a good recovery rate, as shown in Table 4.

[0057] Table 4 Sample recovery results

[0058] Step (4): Determination of inulin-type fructan content in Codonopsis pilosula samples For the 20 batches of Codonopsis samples collected, including 13 batches of Codonopsis produced in Shanxi (numbered 1-13) and 7 batches of Codonopsis produced in Gansu (numbered 14-20), 3 portions of each batch were taken to prepare the test solution. Preparation of the test solution of total polysaccharides from Codonopsis produced in Shanxi: accurately weigh 1.0 mg of Codonopsis polysaccharide powder and dissolve it in 1.0 mL of ultrapure water to obtain a 1 mg / mL test solution; preparation of the test solution of total polysaccharides from Codonopsis produced in Gansu: accurately weigh 2.5 mg of Codonopsis polysaccharide powder and dissolve it in 1.0 mL of ultrapure water to obtain a 2.5 mg / mL test solution. HPGPC analysis was performed according to the above chromatographic conditions, and the peak area of ​​the chromatographic peak at the corresponding position of the reference substance was recorded and its content was calculated. The results showed that the average percentage of inulin-type fructan in Codonopsis pilosula produced in Shanxi and Gansu were 8.4±3.1% and 3.5±0.36%, respectively. The difference in content was statistically significant. The results are shown in Table 5, indicating the feasibility of this method.

[0059] Table 5 Detection results of inulin-type fructan content in 20 batches of Codonopsis pilosula 3. Specific embodiments Example 1

[0060] A method for determining the inulin-type fructan content in Codonopsis pilosula (No. 9 in Table 5) is as follows: Step (1), extraction of total polysaccharides from Codonopsis pilosula.

[0061] Take 100 g of Codonopsis pilosula, extract it with methanol-ethyl acetate extract (volume ratio 1:0.2) at a material-liquid ratio of 1:5, filter out the residue, collect the methanol-ethyl acetate extract and recover the methanol-ethyl acetate.

[0062] The residue was extracted with distilled water at a solid-liquid ratio of 1:5, and the extraction was performed 3 times. The 3 extractions were combined and concentrated to 1 / 4 volume under reduced pressure, precipitated with 70% ethanol, centrifuged at 1500 rpm for 15 minutes, the supernatant was removed, and the precipitate was washed with ether, ethyl acetate, acetone, and anhydrous ethanol for 3 times in sequence, and freeze-dried to obtain a total polysaccharide sample of Codonopsis pilosula, which weighed 20.7 g.

[0063] Step (2): Preparation of test solution Preparation of the test solution of total polysaccharides from Codonopsis pilosula: Accurately weigh 1.0 mg of Codonopsis pilosula polysaccharide powder and dissolve it in 1.0 mL of ultrapure water to obtain a 1 mg / mL test solution.

[0064] 3. Determination of inulin-type fructan content in Codonopsis samples HPGPC analysis was performed under the following chromatographic conditions.

[0065] HPGPC chromatographic conditions: The chromatographic conditions were Agilent 1260 HPLC system, chromatographic column: TSK gel G4000PWXL (7.8mm×30cm, 10μm); detector: differential refractometer (1260 RID-G1362A); mobile phase: ultrapure water; flow rate 0.3ml / min; column temperature: 35℃; detector temperature: 35℃; injection volume 10.0μL, recorded peak area 265054.03.

[0066] Substituting the peak area into the standard curve equation Y=6.27×107X-3545.75, the mass of inulin-type fructan in 10μL injection volume was calculated to be 4.28μg.

[0067] Then, the mass of inulin-type fructan in total polysaccharides is calculated as: (4.28 / 10)×1000×1000×20.7=8.9g; Then, the percentage of inulin-type fructan in the Codonopsis pilosula medicinal material in this Example 1 is calculated as: 8.9 / 100×100%=8.9%. Example 2

[0068] A method for determining the inulin-type fructan content in Codonopsis pilosula (No. 6 in Table 5) is as follows: Step (1), extraction of total polysaccharides from Codonopsis pilosula.

[0069] Take 100 g of Codonopsis pilosula, extract it with methanol-ethyl acetate extract (1:0.3) at a solid-liquid ratio of 1:8, filter out the residue, collect the methanol-ethyl acetate extract and recover the methanol-ethyl acetate.

[0070] The residue was extracted with distilled water at a solid-liquid ratio of 1:8, and the extraction was performed 3 times. The 3 extractions were combined and concentrated to 1 / 4 volume under reduced pressure, precipitated with 80% ethanol, centrifuged at 1500 rpm for 15 minutes, the supernatant was removed, and the precipitate was washed 3 times with ether, ethyl acetate, acetone, and anhydrous ethanol in sequence, and freeze-dried to obtain a total polysaccharide sample of Codonopsis pilosula, which weighed 23.5 g.

[0071] Step (2): Preparation of test solution Preparation of the test solution of total polysaccharides from Codonopsis pilosula: Accurately weigh 1.0 mg of Codonopsis pilosula polysaccharide powder (prepared in step (1)) and dissolve it in 1.0 mL of ultrapure water to obtain a 1 mg / mL test solution.

[0072] Step (3): Determination of inulin-type fructan content in Codonopsis pilosula samples HPGPC analysis was performed under the following chromatographic conditions.

[0073] HPGPC chromatographic conditions: The chromatographic conditions were Agilent 1260 HPLC system, chromatographic column: TSK gel G4000PWXL (7.8mm×30cm, 10μm); detector: differential refractometer (1260 RID-G1362A); mobile phase: ultrapure water; flow rate 0.3ml / min; column temperature: 35℃; detector temperature: 35℃; injection volume 10.0μL, recorded peak area 198664.60.

[0074] Substituting the peak area into the standard curve equation Y=6.27×107X-3545.75, the mass of inulin-type fructan in 10μL injection volume was calculated to be 3.23μg.

[0075] Then, the mass of inulin-type fructan in total polysaccharides is calculated as: 3.23 / 10×1000×1000×23.5=7.6g; Then, the percentage of inulin-type fructan in the Codonopsis pilosula medicinal material in this Example 2 is calculated as: 7.6 / 100×100%=7.6%. Example 3

[0076] A method for determining the inulin-type fructan content in Codonopsis pilosula (No. 16 in Table 5) is as follows: Step (1), extraction of total polysaccharides from Codonopsis pilosula Take 100 g of Codonopsis pilosula, extract it with methanol-ethyl acetate extract (1:0.5) at a solid-liquid ratio of 1:10, filter out the residue, collect the methanol-ethyl acetate extract and recover the methanol-ethyl acetate.

[0077] The residue was extracted with distilled water at a solid-liquid ratio of 1:10, and the extraction was performed 3 times. The 3 extractions were combined and concentrated to 1 / 4 volume under reduced pressure, precipitated with 80% ethanol, centrifuged at 1500 rpm for 15 minutes, the supernatant was removed, and the precipitate was washed 3 times with ether, ethyl acetate, acetone, and anhydrous ethanol in sequence, and freeze-dried to obtain a total polysaccharide sample of Codonopsis pilosula, which weighed 13.4 g.

[0078] (2) Preparation of test solution Preparation of the test solution of total polysaccharides from Codonopsis pilosula: Accurately weigh 2.5 mg of Codonopsis pilosula polysaccharide powder (prepared in step (1)) and dissolve it in 1.0 mL of ultrapure water to obtain a 2.5 mg / mL test solution.

[0079] Step (3): Determination of inulin-type fructan content in Codonopsis pilosula samples HPGPC analysis was performed under the following chromatographic conditions.

[0080] HPGPC chromatographic conditions: The chromatographic conditions were Agilent 1260 HPLC system, chromatographic column: TSK gel G4000PWXL (7.8mm×30cm, 10μm); detector: differential refractometer (1260 RID-G1362A); mobile phase: ultrapure water; flow rate 0.3ml / min; column temperature: 35℃; detector temperature: 35℃; injection volume 10.0μL, recorded peak area 468064.5.

[0081] Substituting the peak area into the standard curve equation Y=6.27×107X-3545.75, the mass of inulin-type fructan in 10μL injection volume was calculated to be 7.5μg.

[0082] Then, the mass of inulin-type fructan in total polysaccharides is calculated as: 7.5 / 10×1000 / 2.5×1000×13.4=4.0g; Then, the percentage of inulin-type fructan in the Codonopsis pilosula medicinal material in this Example 3 is calculated as: 4 / 100×100%=4%.

[0083] The above is only a specific implementation of the present invention, which enables those skilled in the art to understand or implement the present invention. Although detailed descriptions are given with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments, and they should all be covered by the protection scope of the claims.

Claims

1. A method for determining the content of inulin-type fructan in Codonopsis pilosula, characterized in that: The steps include: Step (1), preparation of total polysaccharides from Codonopsis pilosula; Step (2), preparation of Codonopsis pilosula inulin-type fructan reference substance; Step (3), establishing a linear curve equation of the reference substance mass-chromatographic peak area by HPGPC combined with a differential detector; Step (4), determining the inulin-type fructan content in Codonopsis pilosula from different origins.

2. The method for determining the content of inulin-type fructan in Codonopsis pilosula according to claim 1, wherein: Step (1) specifically comprises: extracting Codonopsis pilosula with a methanol-ethyl acetate extract, filtering to obtain a medicinal residue, collecting the methanol-ethyl acetate extract and recovering the methanol-ethyl acetate; The residue was extracted by reflux extraction with distilled water, concentrated to 1 / 4 to 1 / 3 of the volume by reduced pressure, and then precipitated with ethanol. The supernatant was removed by centrifugation, and the precipitate was washed with ether, ethyl acetate, acetone, and anhydrous ethanol in sequence, and finally freeze-dried to obtain a total polysaccharide sample of Codonopsis pilosula, which was weighed and the weight was recorded.

3. The method for determining the content of inulin-type fructan in Codonopsis pilosula according to claim 2, wherein: In step (1), the volume ratio of methanol to ethyl acetate in the methanol-ethyl acetate extract is 1:0.2 to 1:0.5; the solid-liquid ratio of Codonopsis pilosula to the methanol-ethyl acetate extract is 1:5 to 1:

8.

4. The method for determining the content of inulin-type fructan in Codonopsis pilosula according to claim 3, wherein: In step (1), the solid-liquid ratio of the medicinal residue to distilled water is 1:5 to 1:10; the ethanol used is 60% to 90% ethanol; and the centrifugal speed is 1000 to 1500 rpm.

5. The method for determining the content of inulin-type fructan in Codonopsis pilosula according to claim 1, wherein: Step (2) is specifically as follows: the total polysaccharide of Codonopsis pilosula is dissolved in ethanol, and then ultrafiltration is repeated for multiple times through a 5000Da molecular weight ultrafiltration membrane, the speed of the peristaltic pump is 10 to 20 rpm, and the filtrate is detected by high performance gel permeation chromatography. When a single chromatographic peak is detected, it is determined that the inulin-type fructan reference substance is completely prepared, and the ultrafiltrate is concentrated and freeze-dried to obtain the Codonopsis pilosula inulin-type fructan reference substance.

6. The method for determining the content of inulin-type fructan in Codonopsis pilosula according to claim 5, wherein: In step (2), 30% to 60% ethanol is used and dissolved to 50 mg / L to 100 mg / L.

7. The method for determining the content of inulin-type fructan in Codonopsis pilosula according to claim 6, wherein: In step (2), the reference substance of Codonopsis pilosula inulin type fructan is 1 H-NMR, 13 C-NMR, DEPT identification, using 1 The degree of polymerization was determined by H-NMR.

8. The method for determining the content of inulin-type fructan in Codonopsis pilosula according to claim 1, wherein: Step (3) is specifically as follows: accurately weigh 1.0 mg of Codonopsis pilosula inulin-type fructan reference substance and dissolve it in 1.0 mL of ultrapure water to obtain a 1 mg / mL reference substance solution; The reference solution was accurately aspirated and filtered using a 0.45 μm microporous filter membrane; 10.0 μL, 8.0 μL, 6.0 μL, 4.0 μL, and 2.0 μL were injected for HPGPC analysis, and the peak area and retention time were recorded. The standard curve was drawn with mass X and peak area Y, and the linear regression equation Y=6.27×10 7 X-3545.75, R 2 =0.99994.

9. The method for determining the content of inulin-type fructan in Codonopsis pilosula according to claim 8, wherein: HPGPC chromatographic conditions: The chromatographic conditions were Agilent 1260 HPLC system, chromatographic column: 7.8 mm × 30 cm, 10 μm; detector: differential refractometer; mobile phase: ultrapure water; flow rate 0.3 ml / min; column temperature: 35 °C; detector temperature: 35 °C; injection volume 10.0 μL.

10. The method for determining the content of inulin-type fructan in Codonopsis pilosula according to claim 1, characterized in that: Step (4) specifically comprises taking 3 portions of each batch of the Codonopsis pilosula samples to be tested to prepare the test solution; then performing HPGPC analysis, recording the peak area of ​​the chromatographic peak at the corresponding position of the reference substance and calculating its content; The preparation method of the test solution is as follows: 1) The Codonopsis pilosula sample to be tested is extracted with methanol-ethyl acetate extract at a solid-liquid ratio of 1:5 to 1:8, the drug residue is filtered, the methanol-ethyl acetate extract is collected and the methanol-ethyl acetate is recovered; wherein the ratio of methanol to ethyl acetate in the methanol-ethyl acetate extract is 1:0.2 to 1:0.5; 2) The drug residue is reflux extracted with distilled water at a solid-liquid ratio of 1:5 to 1:10, extracted 3 times, the 3 extractions are combined, concentrated to 1 / 4 to 1 / 3 of the volume under reduced pressure, precipitated with 60% to 90% ethanol, centrifuged at 1000 to 1500 rpm for 15 to 30 minutes, the supernatant is removed, and the precipitate is washed 3 times with ether, ethyl acetate, acetone, and anhydrous ethanol in sequence, freeze-dried to obtain a Codonopsis pilosula total polysaccharide sample, and weighed and recorded the weight; 3) Accurately weigh the Codonopsis pilosula total polysaccharide sample, dissolve it in ultrapure water, and obtain the test solution; HPGPC chromatographic conditions: The chromatographic conditions were Agilent 1260 HPLC system, chromatographic column: 7.8 mm × 30 cm, 10 μm; detector: differential refractometer; mobile phase: ultrapure water; flow rate 0.3 ml / min; column temperature: 35 °C; detector temperature: 35 °C; injection volume 10.0 μL.

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