A method for determining the contents of seven cyclic peptide components in Pseudostellaria heterophylla by one-test-multiple-evaluation method

Abstract

The present invention relates to the technical field of Chinese medicine component detection, and specifically to a method for determining the content of 7 cyclic peptide components in Radix Pseudostellariae by a one-test-multiple-evaluation method. The method comprises: (1) taking 7 cyclic peptide component reference substances, adding methanol to dissolve them, and obtaining a reference substance solution; (2) taking a Radix Pseudostellariae to be tested, adding a methanol solution to ultrasonically extract, filtering, obtaining a filtrate, and dissolving the residue obtained after drying with a methanol solution, filtering, and obtaining a filtrate to obtain a test solution; (3) injecting the reference substance solution and the test solution into a high performance liquid chromatograph, using Radix Pseudostellariae cyclopeptide B as an internal reference, and calculating the relative correction factor f s / i Calculate the content of the cyclic peptide component to be tested; the mobile phase is: acetonitrile (A); any one of water, 0.1% phosphoric acid aqueous solution, 0.1% formic acid aqueous solution or 20mM ammonium acetate aqueous solution (B). The method of the present invention has good durability, simple steps and high accuracy, and provides a basis for the quality control of Pseudostellaria heterophylla.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine component detection, and particularly to a method for determining the contents of seven cyclic peptide components in Pseudostellaria heterophylla by a one-test-multiple-evaluation method. Background Art

[0002] Pseudostellariae Pseudostellariae, also known as Haier Shen, is the root of Haier Shen, a plant of Caryophyllaceae. It has the effects of tonifying the spleen and lungs, replenishing qi and promoting body fluid, and is clinically used for weak spleen and stomach and loss of appetite. Pseudostellariae Pseudostellariae mainly contains polysaccharides, cyclic peptides, volatile oils, alkaloids, saponins, phenols and other ingredients, and has pharmacological effects such as inhibiting tyrosinase, immunomodulation, lowering blood sugar, anti-inflammatory, anti-cancer, protecting cells, and inhibiting tyrosinase.

[0003] Pseudostellariae Radix is ​​included in the 2020 edition of the Chinese Pharmacopoeia, but there is only qualitative authenticity identification, and no qualified content determination indicators. Cyclopeptide components are considered to be characteristic components of Pseudostellariae Radix and one of the main active ingredients. There are as many as 15 types, but the content of each cyclopeptide component varies greatly from place to place. It is difficult to comprehensively evaluate the quality status of Pseudostellariae Radix with only a single cyclopeptide component.

[0004] Quantitative analysis of multicomponents by single marker (QAMS) refers to the use of the intrinsic functional relationship and proportional relationship of the effective ingredients of traditional Chinese medicine to measure only one component (the reference material is easy to obtain) to achieve the simultaneous determination of multiple components (the reference material is difficult to obtain or supply). It is a multi-index quality evaluation model suitable for the characteristics of traditional Chinese medicine. In the case of insufficient reference materials, the use of relative correction factors (RCF) to control the quality of medicinal materials is convenient, fast and cheap. This method will be the development direction of simultaneous quantification of multi-components of traditional Chinese medicine.

[0005] At present, there are few literatures on evaluating the quality of Chinese medicinal materials using the one-test-multiple-evaluation method, and there are no reports on simultaneously determining the contents of multiple components in Pseudostellaria heterophylla using the one-test-multiple-evaluation method.

[0006] Therefore, it is very necessary to develop a one-test-multiple-evaluation method to determine the content of 7 cyclic peptide components in Pseudostellaria heterophylla that can solve the above technical problems. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for determining the contents of 7 cyclic peptide components in Radix Pseudostellariae by a one-test-multiple-evaluation method. The method uses Radix Pseudostellariae cyclic peptide B as an internal reference, can quickly detect the contents of Radix Pseudostellariae cyclic peptide A, Radix Pseudostellariae cyclic peptide A, Radix Pseudostellariae cyclic peptide B, Radix Pseudostellariae cyclic peptide D, Radix Pseudostellariae cyclic peptide E and Radix Pseudostellariae cyclic peptide H in Radix Pseudostellariae, has a high linear correlation coefficient, good precision, repeatability and stability, good sample recovery effect, good durability, simple steps and high accuracy, and provides a basis for quality control of Radix Pseudostellariae.

[0008] The present invention is achieved through the following technical solutions:

[0009] The method for determining the contents of 7 cyclic peptide components in Radix Pseudostellariae by one-test-multiple-evaluation method comprises the following steps:

[0010] (1) Preparation of reference solution: Take 7 kinds of cyclopeptide component reference substances, namely, Radix Pseudostellariae Cyclic Peptide A, Radix Pseudostellariae Cyclic Peptide B ... D, Radix Pseudostellariae Cyclic Peptide E and Radix Pseudostellariae Cyclic Peptide H, add methanol to dissolve them, and obtain reference solution;

[0011] (2) Preparation of test solution: Take the Radix Pseudostellariae Radix to be tested, add methanol solution to ultrasonic extraction, filter, take the filtrate, add methanol solution to the residue obtained after drying, filter, take the filtrate to obtain the test solution;

[0012] (3) Inject the reference solution and the test solution into the HPLC, use Pseudostigma cyclotide B as the internal reference, and calculate the relative correction factor f s / i Calculate the contents of the other 6 cyclic peptide components to be tested;

[0013] The mobile phase used in the high performance liquid chromatograph in step (3) is: phase A, acetonitrile; phase B, any one of water, 0.1% phosphoric acid aqueous solution, 0.1% formic acid aqueous solution or 20 mM ammonium acetate aqueous solution, that is, the mobile phase is any one of the following: ① acetonitrile (A)-water (B); ② acetonitrile (A)-0.1% phosphoric acid aqueous solution (B); ③ acetonitrile (A)-0.1% formic acid aqueous solution (B); ④ acetonitrile-20 mM ammonium acetate aqueous solution.

[0014] In some embodiments of the present invention, the concentrations of the seven cyclic peptide component reference substances in the reference substance solution in step (1) are all 10 μg / mL-300 μg / L.

[0015] In the preparation process of the reference substance solution of the present invention, a single reference substance solution may be prepared first and then mixed, or the reference substance solution may be directly prepared, and the former is preferred.

[0016] In some preferred embodiments of the present invention, the preparation of the reference solution includes: taking Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix D, Pseudostellariae Radix E and Pseudostellariae Radix H reference substances, respectively dissolving and diluting with methanol to obtain a single reference solution with a concentration of 0.3 mg / mL-3 mg / mL; placing the single reference solution in a volumetric flask, diluting with methanol solution to obtain a reference solution with a concentration of 10 μg / mL-300 μg / mL.

[0017] In some embodiments of the present invention, step (2) further comprises at least one of the following conditions:

[0018] ii1) the ratio of the Pseudostellaria heterophylla sample to be tested to the methanol solution is 2-5 g: 5-10 mL;

[0019] ii2) the volume percentage concentration of the methanol solution used in the ultrasonic extraction is 60%-100%;

[0020] ii3) The ultrasonic extraction time is 0.5h-1.5h.

[0021] In step (3) of the present invention, the peak areas of Pseudostellariae Cyclic Peptide B in the test solution and the reference solution are respectively determined by high performance liquid chromatography, and the content of Pseudostellariae Cyclic Peptide B is calculated using an external standard one-point method or a working curve method.

[0022] In some embodiments of the present invention, the elution method adopted by the high performance liquid chromatograph in step (3) is gradient elution, which specifically includes any one of the following:

[0023] ①0-5min, 20%A; 5-15min, 20%→40%A; 15-30min, 40%A; 30-36min, 40%→80%A; 36-55min, 80%A; 55-56min, 80%→20%A;

[0024] ②0-5min, 2%→35%A; 5-20min, 35%→45%A; 20-25min, 45%→55%A; 25-30min, 55%→80%A, 30-40min, 80%A;

[0025] ③0-5min, 30%A; 5-60min, 30%→45%A; 60-61min, 45%→80%A; 61-70min, 80%A;

[0026] ④0-15min, 20%→30%A; 15-35min, 30%→40%A; 35-45min, 40%A; 45-55min, 40%→80%A; 55-65min, 80%A.

[0027] In some embodiments of the present invention, the test conditions of the high performance liquid chromatograph in step (3) include:

[0028] Chromatographic column: Select a chromatographic column with a packing size of 5 μm;

[0029] Flow rate: 0.8-1.2mL / min;

[0030] Column temperature: 25-35°C;

[0031] Injection volume: 2-25 μL;

[0032] Detection wavelength: 203nm-220nm.

[0033] In some embodiments of the present invention, the test conditions of the high performance liquid chromatograph in step (3) include:

[0034] Chromatographic column: YMC Triart C 18 ;

[0035] Mobile phase: Phase A, acetonitrile; Phase B, water;

[0036] Gradient elution program: 0-5 min, 20% A; 5-15 min, 20%→40% A; 15-30 min, 40% A; 30-36 min, 40%→80% A; 36-55 min, 80% A; 55-56 min, 80%→20% A;

[0037] Flow rate: 0.8-1.2mL / min;

[0038] Column temperature: 25-35°C;

[0039] Injection volume: 5-25 μL;

[0040] Detection wavelength: 203-220nm.

[0041] The invention adopts relative retention time and relative correction factor to respectively determine and calculate the peak positions and contents of Pseudostellariae Radix A, Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix D, Pseudostellariae Radix E and Pseudostellariae Radix H in the sample.

[0042] In some embodiments of the present invention, the relative retention times of Pseudostellariae cyclopeptide A, Pseudostellariae cyclopeptide A, Pseudostellariae cyclopeptide B, Pseudostellariae cyclopeptide D, Pseudostellariae cyclopeptide E and Pseudostellariae cyclopeptide H relative to Pseudostellariae cyclopeptide B are 1.07-1.38, 0.61-0.87, 0.70-0.99, 0.87-1.23, 1.25-1.74, 1.14-1.53, respectively.

[0043] In some preferred embodiments of the present invention, the relative retention times of Pseudostellariae cyclopeptide A, Pseudostellariae cyclopeptide A, Pseudostellariae cyclopeptide B, Pseudostellariae cyclopeptide D, Pseudostellariae cyclopeptide E and Pseudostellariae cyclopeptide H relative to Pseudostellariae cyclopeptide B are 1.21, 0.75, 0.87, 1.09, 1.50 and 1.33 respectively.

[0044] In some embodiments of the present invention, in step (3), the reference solution and the test solution are injected into a high performance liquid chromatograph, and the peak areas of the seven cyclic peptide components are recorded according to the liquid chromatogram obtained from the reference solution. The relative correction factors f of the other six cyclic peptide components to be tested are calculated by using a multi-point calibration method with Pseudostigma cyclotide B as the internal reference. s / i , according to f s / i Calculate the contents of the other 6 cyclic peptide components in the sample to be tested.

[0045] In some embodiments of the present invention, the relative correction factor f in step (3) s / i The multi-point correction method is used for calculation, and the calculation method is as follows:

[0046] Relative correction factor f s / i =f s / f i =(C i ×A s ) / (C s ×A i );

[0047] Among them, f i is the correction factor for the cyclic peptide component to be tested in the reference solution, C i is the content of the cyclic peptide component to be tested in the reference solution, A i is the peak area of ​​the cyclic peptide component to be tested in the reference solution, f s is the internal reference correction factor in the reference solution, C s is the content of internal reference substance in the reference solution, A s is the peak area of ​​the internal reference substance in the reference solution.

[0048] In some preferred embodiments of the present invention, the relative correction factor f of Pseudospermum officinale A, Pseudospermum officinale A, Pseudospermum officinale B, Pseudospermum officinale D, Pseudospermum officinale E and Pseudospermum officinale H relative to Pseudospermum officinale B is s / i They are 1.11-1.62, 0.82-1.20, 0.73-1.18, 0.78-1.34, 0.81-1.35 and 0.58-1.25 respectively.

[0049] In some preferred embodiments of the present invention, the relative correction factors of Pseudostellariae Radix A, Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix D, Pseudostellariae Radix E and Pseudostellariae Radix H relative to Pseudostellariae Radix B are 1.42, 0.99, 1.01, 0.99, 1.12 and 0.89 respectively.

[0050] In some embodiments of the present invention, the content of the cyclic peptide component to be tested in step (3) is calculated as follows:

[0051] K x =f s / i ×(K s ×T x / T s );

[0052] Among them, T x and T s are the peak areas of the cyclic peptide component to be tested and the internal reference in the test solution, respectively. x and K s are the contents of the cyclic peptide component to be tested and the internal reference substance in the test solution, respectively. s / i is the relative correction factor of the cyclic peptide component to be measured.

[0053] The beneficial effects of the present invention are:

[0054] The present invention creates an accurate and low-cost quality control and evaluation method for Radix Pseudostellariae. The contents of seven cyclopeptide components, namely, Radix Pseudostellariae cyclopeptide A, Radix Pseudostellariae cyclopeptide B, Radix Pseudostellariae cyclopeptide A, Radix Pseudostellariae cyclopeptide B, Radix Pseudostellariae cyclopeptide D, Radix Pseudostellariae cyclopeptide E and Radix Pseudostellariae cyclopeptide H, are simultaneously determined through a one-measurement-multiple-evaluation method. That is, only one reference substance, cyclopeptide B, is needed to calculate the contents of the other six components through relative correction factors.

[0055] The cyclic peptide components in the Radix Pseudostellariae in the present invention have similar structures and are numerous, and are difficult to separate by chromatography. The present invention establishes a mobile phase system of four solvent combinations, and achieves good baseline separation of the cyclic peptide components in the Radix Pseudostellariae through gradient elution, thereby ensuring accurate and reliable positioning of each chromatographic peak in the one-measurement-multiple-evaluation method.

[0056] The invention fills the blank of no index or active ingredient content determination in quality control of Radix Pseudostellariae Radix and provides a scientific basis for solving the quality control problem of Radix Pseudostellariae Radix. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 It is the HPLC chromatogram of the mixed reference substance of Example 1 of the present invention.

[0058] Figure 2 It is the HPLC chromatogram of gradient a in Example 1 of the present invention.

[0059] Figure 3 It is the HPLC chromatogram of gradient b in Example 1 of the present invention.

[0060] Figure 4 It is the HPLC chromatogram of gradient c in Example 1 of the present invention.

[0061] Figure 5 It is the HPLC chromatogram of gradient d in Example 1 of the present invention.

[0062] Figure 6 The HPLC chromatogram of methanol-water as the mobile phase in Example 3 of the present invention.

[0063] Figure 7 This is the HPLC chromatogram of acetonitrile-0.1% phosphoric acid water as the mobile phase in Example 3 of the present invention.

[0064] Figure 8 This is the HPLC chromatogram of acetonitrile-0.1% formic acid aqueous solution as the mobile phase in Example 3 of the present invention.

[0065] Fig. 9 This is the HPLC chromatogram of acetonitrile-20mmol ammonium acetate aqueous solution as the mobile phase in Example 3 of the present invention. DETAILED DESCRIPTION

[0066] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0067] The instruments and materials used in the present invention are as follows:

[0068] instrument:

[0069] Agilent 1260 Infinity II Prim high performance liquid chromatograph (Agilent Technologies, Inc., USA), ThermoUltiMate 3000 high performance liquid chromatograph (Thermo Fisher Scientific, Inc., USA), MSA6.6S-CE electronic balance (Sartorius, Germany), MSA224S electronic balance (Sartorius, Germany), SB-500DTY ultrasonic cleaning machine (Ningbo Xinzhi Biotechnology Co., Ltd.), HH-4 digital display constant temperature water bath (Changzhou Guohua Electric Appliance Co., Ltd.).

[0070] Material:

[0071] Reference substances: Pseudostellariae cyclopeptide A (batch number: P220127-T250, content ≥ 95%), Pseudostellariae cyclopeptide B (batch number: HS20186-2-0721-A, content ≥ 95%), Pseudostellariae cyclopeptide D (batch number: HS20186-4-0721-A, content ≥ 95%), Pseudostellariae cyclopeptide E (batch number: HS20186-7-0721-A, content ≥ 95%), Pseudostellariae cyclopeptide H (batch number: HS20186-6-0721-A, content ≥ 95%), Pseudostellariae cyclopeptide A (batch number: HS20186-5-0721-A, content ≥ 95%) were all purchased from Kunming Dingzhong Scientific Instrument Co., Ltd.; Pseudostellariae cyclopeptide B (batch number: 145459-19-4, content ≥ 95%, purchased from Shanghai Standard Company).

[0072] Reagents: Methanol and acetonitrile were of chromatographic grade (Merck, Germany); water was ultrapure water.

[0073] Samples: 10 batches of Pseudostellariae Radix samples were from Tianren Pharmaceutical Co., Ltd. (Batch numbers: 220727001, 220727002, 220727003, 220727004, 220727005, 220813001, 220813002, 220813003, 220813004, 220813005).

[0074] The present invention provides a quality evaluation method of Radix Pseudostellariae Radix based on one-test-multiple-evaluations, comprising the following steps:

[0075] 1) Preparation of single reference substance solution: Take Pseudostellariae Radix Cyclotide A, Pseudostellariae Radix Cyclotide B, Pseudostellariae Radix Cyclotide A, Pseudostellariae Radix Cyclotide B, Pseudostellariae Radix Cyclotide D, Pseudostellariae Radix Cyclotide E and Pseudostellariae Radix Cyclotide H, respectively, dissolve and dilute with methanol to obtain single reference substance stock solution;

[0076] 2) Preparation of mixed reference substance solution: Take the single reference substance stock solutions obtained in step 1), place them in volumetric flasks, and dilute them with methanol solution to obtain mixed reference substance solutions;

[0077] 3) Take a sample, add methanol solution for ultrasonic treatment, filter, concentrate the filtrate to near dryness, add methanol solution to dissolve, transfer to a volumetric flask, make up to volume, shake well, filter, take the filtrate to obtain the test solution;

[0078] 4) injecting the mixed reference solution obtained in step 2) into a high performance liquid chromatograph, injecting and measuring, obtaining a liquid chromatogram, recording the peak area of ​​each component, and using a multi-point calibration method, taking Pseudostellariae cyclotide B as an internal reference, calculating the relative calibration factors of the other six cyclotide components;

[0079] 5) Analyze and determine the basis for the chromatographic peak positioning of the cyclic peptide component to be tested.

[0080] Chromatographic conditions:

[0081] Chromatographic column: A chromatographic column with a filler size of 5 μm was selected; the mobile phase was selected from the following: ① acetonitrile (A)-water (B); ② acetonitrile (A)-0.1% phosphoric acid aqueous solution (B); ③ acetonitrile (A)-0.1% formic acid aqueous solution (B); ④ acetonitrile-20 mM ammonium acetate aqueous solution, for gradient elution; flow rate: 1.0 mL / min; detection wavelength: 203 nm; injection volume: 20 μL. According to the chromatographic conditions of Example 1, the peaks in the sample chromatogram were well separated, and the theoretical plate number was greater than 3000 based on Pseudostigma cyclotide B.

[0082] The gradient elution program uses one of the following elution schemes:

[0083] Gradient a: 0-5min, 20%A; 5-15min, 20%→40%A; 15-30min, 40%A; 30-36min, 40%→80%A; 36-55min, 80%A; 55-56min, 80%→20%A.

[0084] Gradient b: 0-5 min, 2%→35% A; 5-20 min, 35%→45% A; 20-25 min, 45%→55% A; 25-30 min, 55%→80% A, 30-40 min, 80% A.

[0085] Gradient c: 0-5 min, 30% A; 5-60 min, 30%→45% A; 60-61 min, 45%→80% A; 61-70 min, 80% A.

[0086] Gradient d: 0-15min, 20%→30%A; 15-35min, 30%→40%A; 35-45min, 40%A; 45-55min, 40%→80%A; 55-65min, 80%A.

[0087] Example 1

[0088] Step 1) Preparation of single reference solution:

[0089] Take appropriate amount of Pseudostellariae Radix Cyclotide A, Pseudostellariae Radix Cyclotide B, Pseudostellariae Radix Cyclotide A, Pseudostellariae Radix Cyclotide B, Pseudostellariae Radix Cyclotide D, Pseudostellariae Radix Cyclotide E and Pseudostellariae Radix Cyclotide H reference substances, accurately weigh them, add methanol to dissolve and dilute them into single reference substance stock solutions with mass concentrations of 1.4546, 1.3160, 1.5021, 1.2704, 1.2849, 1.3754 and 1.2832 mg / mL respectively.

[0090] Step 2) Preparation of mixed reference solution:

[0091] Take an appropriate amount of the single reference substance stock solution of step 1), place it in a 25mL volumetric flask, add 80% methanol solution to dilute it into a mixed reference substance solution I with mass concentrations of 116.367, 105.280, 120.171, 101.632, 102.791, 110.030, and 102.658μg / mL for Radix Pseudostellariae Cyclic Peptide A, Radix Pseudostellariae Cyclic Peptide B, Radix Pseudostellariae Cyclic Peptide D, Radix Pseudostellariae Cyclic Peptide E, and Radix Pseudostellariae Cyclic Peptide H, respectively, and further dilute the mixed reference substance solution with concentrations of 2 times, 5 times, 10 times, 20 times, and 100 times (for linear determination). The HPLC chromatogram of the mixed reference substance is shown in Figure 1 .

[0092] Step 3) Preparation of test solution:

[0093] Take about 4 g of Pseudostellariae Radix sample, weigh it accurately, put it in a stoppered conical flask, accurately add 100 mL of 80% methanol solution, extract by ultrasonic for 60 min, cool, filter, concentrate the filtrate to near dryness, add 20% methanol solution to dissolve, transfer to a 5 mL volumetric flask, make up to volume, shake well, filter, take the filtrate to obtain the test solution.

[0094] Step 4) Chromatographic conditions:

[0095] The chromatographic column was YMC-Triart C18 (4.6 mm × 250 mm, 5 μm), flow rate: 1.0 mL / min; detection wavelength: 203 nm; column temperature: 35 °C; injection volume: 20 μL, mobile phase was acetonitrile (A)-water (B), and the gradient elution program selected one of the following elution schemes:

[0096] Gradient a: 0-5 min, 20% A; 5-15 min, 20%→40% A; 15-30 min, 40% A; 30-36 min, 40%→80% A; 36-55 min, 80% A; 55-56 min, 80%→20% A; the HPLC chromatogram of gradient a is shown in Figure 2 Unless otherwise specified, the investigation tests in Example 2 and Example 3 were conducted under the chromatographic conditions of gradient a.

[0097] Gradient b: 0-5 min, 2%→35% A; 5-20 min, 35%→45% A; 20-25 min, 45%→55% A; 25-30 min, 55%→80% A, 30-40 min, 80% A; HPLC chromatogram of gradient b is shown in Figure 3 .

[0098] Gradient c: 0-5 min, 30% A; 5-60 min, 30%→45% A; 60-61 min, 45%→80% A; 61-70 min, 80% A; HPLC chromatogram of gradient c is shown in Figure 4 .

[0099] Gradient d: 0-15 min, 20%→30% A; 15-35 min, 30%→40% A; 35-45 min, 40% A; 45-55 min, 40%→80% A; 55-65 min, 80% A. The HPLC chromatogram of gradient d is shown in Figure 5 .

[0100] Example 2

[0101] 2.1 Investigation of linear relationship

[0102] 20 μL of the mixed reference solution of step 2) in Example 1 was accurately pipetted and injected into a liquid chromatograph. The sample was injected and measured according to the chromatographic conditions of Example 1. The logarithm of the mass concentration of the reference substance was used as the abscissa (X) and the logarithm of the peak area was used as the ordinate (Y). A standard curve was drawn. The results are shown in Table 1. The linear correlation coefficients (r) of the seven components were all greater than 0.999, indicating that the linear relationship of each cyclic peptide component was good within the corresponding mass range.

[0103] Table 1 Regression equations, correlation coefficients, and linear ranges of the seven cyclic peptide components

[0104]

[0105] 2.2 Precision test

[0106] Accurately pipette 20 μL of the mixed reference solution I of step 2) in Example 1, and continuously inject 6 times according to the chromatographic conditions of Example 1, and the RSD% of the peak areas of Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix D, Pseudostellariae Radix E and Pseudostellariae Radix G were measured to be 0.039%, 0.038%, 0.022%, 0.020%, 0.029%, 0.048% and 0.036%, respectively, indicating that the instrument has good precision.

[0107] 2.3 Repeatability test

[0108] Take about 4g of the same batch of sample powder, and make 6 parallel portions, accurately weigh them, prepare the test solution according to the method of step 3) in Example 1, and determine it according to the chromatographic conditions of Example 1. The relative retention time RSDs of Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix D, Pseudostellariae Radix E and Pseudostellariae Radix H were 0.01%, 0.0%, 0.02%, 0.02%, 0.02%, 0.01% and 0.01% respectively; the content RSDs were 1.1%, 1.2%, 1.7%, 3.4%, 3.1%, 3.5% and 3.5% respectively, and the RSDs were in line with the requirements of the pharmacopoeia, indicating that the method has good repeatability.

[0109] 2.4 Stability test

[0110] Take the same portion of the test solution as in the repeatability test, and measure the sample at room temperature at 0, 2, 4, 8, 12, 24, and 48 h according to the chromatographic conditions of Example 1. The relative retention time RSDs of Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix D, Pseudostellariae Radix E and Pseudostellariae Radix H were 0.2%, 0.0%, 0.1%, 0.1%, 0.2%, 0.3% and 0.2%, respectively; the peak area RSDs were 3.0%, 0.18%, 2.7%, 3.9%, 1.9%, 1.4% and 2.2%, respectively. The RSDs were in compliance with the requirements of the pharmacopoeia, indicating that the test solution had good stability within 48 h.

[0111] 2.5 Sample recovery test

[0112] Take 9 portions of sample powder with known content, each portion is about 2g, accurately weighed, and placed in stoppered conical flasks respectively, and accurately add low concentration (50%), medium concentration (100%), and high concentration (150%) of Radix Pseudostellariae Cyclic Peptide A, Radix Pseudostellariae Cyclic Peptide B, Radix Pseudostellariae Cyclic Peptide A, Radix Pseudostellariae Cyclic Peptide B, Radix Pseudostellariae Cyclic Peptide D, Radix Pseudostellariae Cyclic Peptide E and Radix Pseudostellariae Cyclic Peptide H reference solution, prepare the test sample spiked solution in parallel according to the method of step 3) of Example 1, and determine according to the chromatographic conditions of Example 1, and calculate the recovery rate of each cyclic peptide component. The average recoveries of Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix D, Pseudostellariae Radix E and Pseudostellariae Radix H were 101.9%, 99.8%, 99.0%, 97.8%, 92.2%, 94.6% and 96.9%, respectively, and the RSDs were 2.4%, 2.3%, 1.5%, 2.7%, 2.3%, 2.4% and 2.9%, respectively, as shown in Table 2.

[0113] Table 2 Results of sample recovery test (n=9)

[0114]

[0115] 2.6 Calculation of relative correction factors for seven cyclic peptide components

[0116] Accurately pipette the mixed reference solution of different concentrations under step 2) of Example 1, inject 20 μL of each according to the chromatographic conditions of Example 1, record the peak area of ​​each component, and use the multi-point calibration method to calculate the relative correction factors f of the other 6 cyclic peptide components to be tested with Pseudostigma cyclotide B as the internal reference. s / i , the calculation formula is f s / i =f s / f i =(C i ×A s ) / (C s ×A i ), where C i is the content of the cyclic peptide component to be tested, A i is the peak area of ​​the cyclic peptide component to be tested, C s is the internal reference content, A s is the peak area of ​​the internal reference, the results are shown in Table 3, the six components f s / i The RSDs of the values ​​were all less than 2%, indicating that the method was stable and had good repeatability.

[0117] Table 3 Results of relative correction factors for each component

[0118]

[0119] 2.7 One-test-multiple-evaluation system durability assessment

[0120] In the process of establishing the one-measurement-multiple-evaluation method, all factors that cause changes in the response value of the chromatographic system detector may cause fluctuations in the relative correction factor, thereby introducing errors between the calculated value and the measured value. For this reason, the durability of the system was investigated using four factors: different instruments, chromatographic columns, column temperatures, and flow rates, to ensure that the established relative correction factor has good reproducibility.

[0121] 2.7.1 Investigation of different instruments and columns

[0122] Two different chromatographic systems (Agilent 1260 Infinity II Prim high performance liquid chromatograph (Agilent 1260 for short) and Thermo UltiMate 3000 high performance liquid chromatograph (Thermo Fisher U3000 for short)) and two different types of chromatographic columns (column 1: YMC-Triart C18 (250×4.6mm, 5μm); column 2: Agilent SB-C18 (250×4.6mm, 5μm)) were selected to investigate the relative correction factors of each cyclic peptide component, as shown in Table 4. The results showed that there was no significant difference in the influence of different chromatographic instruments and different chromatographic columns on the relative correction factors of each cyclic peptide (RSD was less than 3%).

[0123] Table 4 Effects of different chromatographs and columns on relative correction factors

[0124]

[0125] 2.7.2 Investigation of different column temperatures

[0126] The other chromatographic conditions remained unchanged, and different column temperatures (25, 30, 40°C) were set to determine the relative correction factors of each cyclic peptide, as shown in Table 5. The results showed that there was no significant difference in the effect of different column temperatures on the relative correction factors of each component (RSD was less than 2%).

[0127] Table 5 Effect of different column temperatures on relative correction factors

[0128]

[0129] 2.7.3 Investigation of different flow rates

[0130] The other chromatographic conditions remained unchanged, and different flow rates (0.90, 1.00, 1.10 mL / min) were set to determine the relative correction factors of each cyclic peptide component, as shown in Table 6. The results showed that there was no significant difference in the effect of different flow rates on the relative correction factors of each cyclic peptide component (RSD was less than 2%).

[0131] Table 6 Effect of different flow rates on relative correction factors

[0132]

[0133] Example 3

[0134] 3.1 Positioning of the chromatographic peaks of the six components

[0135] In practical applications, the one-measurement-multiple-evaluation method uses relative correction factors to calculate the content, and does not require reference materials for the components to be measured other than the reference materials. The positioning of the chromatographic peaks of these components is also achieved through reference materials. However, the components of traditional Chinese medicine are complex, and there are other chromatographic peaks in the chromatogram. Therefore, the correct positioning of the chromatographic peaks of the components to be measured is a prerequisite for accurate measurement by the one-measurement-multiple-evaluation method. The present invention uses the relative retention time ratio of the components to be measured and the internal reference material for positioning (i.e., the relative retention value method t i / s =t i / t s , where t i is the retention time of the analyte, t s is the retention time of the internal reference), for this purpose, different chromatographic systems and different chromatographic column conditions were investigated. i / s The results are shown in Table 7. The results show that the relative retention time RSDs of Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix D, Pseudostellariae Radix E and Pseudostellariae Radix H are all less than 2%, which meets the requirements of one-test-multiple-evaluation chromatographic peak positioning.

[0136] Table 7 Effects of different chromatographs and columns on relative retention time

[0137]

[0138] 3.2 Comparison of the results between the one-test-multiple-evaluation method and the external standard method

[0139] Ten batches of Radix Pseudostellariae Radix were prepared into test solutions according to the method of step 3) of Example 1, and the samples were injected and measured according to the chromatographic conditions of Example 1, and the chromatograms were recorded. The contents of each component were calculated by the single measurement multiple evaluation method (QAMS) and the external standard method (ESM), and the results of the two methods were compared by relative deviation (RD). The calculation formula was RD=|QAMS value-ESM value| / ESM value×100%, and the calculation results are shown in Tables 8 and 9.

[0140] The calculation method of the content of the cyclic peptide component to be tested by the one-test-multiple-evaluation method is as follows:

[0141] K x =f s / i ×(K s ×T x / T s );

[0142] Among them, T x and T s are the peak areas of the cyclic peptide component to be tested and the internal reference in the test solution, respectively. x and K s are the contents of the cyclic peptide component to be tested and the internal reference substance in the test solution, respectively. s / i is the relative correction factor of the cyclic peptide component to be measured.

[0143] The results showed that the content determination results obtained by the two methods were in line with the provisions of the pharmacopoeia, and the paired sample T test showed that there was no significant difference between the two methods. The relative correction factor established by the present invention is accurate and reliable, and can be used for the determination and quality evaluation of Pseudostellariae Radix A (HA), Pseudostellariae Radix B (HB), Pseudostellariae Radix A (PA), Pseudostellariae Radix B (PB), Pseudostellariae Radix D (PD), Pseudostellariae Radix E (PE) and Pseudostellariae Radix Glycyrrhizae Radix (PG).

[0144] Table 8 Comparison of sample content determination results by external standard method and one-measurement-multiple-evaluation method (μg / g, n=2)

[0145]

[0146] Table 9 Comparison of sample content determination results by external standard method and one-measurement-multiple-evaluation method (μg / g, n=2)

[0147]

[0148] When the content of multiple index components of traditional Chinese medicine is determined by the one-test-multiple-evaluation method, one of the effective ingredients or index components with clear pharmacological effects is usually used as an internal reference. Pseudostellariae cyclotide is both an active ingredient and a characteristic ingredient. Among the seven cyclotides of Pseudostellariae cyclotide, Pseudostellariae cyclotide B has a stable and high content, so the present invention uses Pseudostellariae cyclotide B as an internal reference.

[0149] 3.3 Optimization of sample pretreatment conditions

[0150] The effects of different extraction methods (ultrasound, heating reflux) on the test results were investigated, and the contents of 7 index components were selected as the objects of investigation. It was found that the contents of cyclic peptide substances were basically the same under different extraction methods, so heating reflux is the preferred extraction method of the present invention.

[0151] 3.4 Selection of extraction time

[0152] The effects of different ultrasonic times (15min, 30min, 45min, 60min, and 90min) on the test results were investigated, and the contents of each cyclic peptide component under different ultrasonic treatment time conditions were compared. The results showed that the content change trends of each component were basically consistent with the change of ultrasonic time, and the results of 60min and 90min were consistent, so the preferred extraction time was 60min.

[0153] 3.5 Selection of extraction solvent concentration

[0154] The effects of different volume percentage concentrations of methanol (10%, 30%, 50%, 80%, 90%) on the test results were investigated, and the contents of each cyclic peptide component under different concentrations of extraction solvent were compared. The results showed that the content of each component had a basically consistent trend with the change of the volume percentage concentration of methanol. When the volume percentage concentration of methanol was between 10% and 50%, the content of each cyclic peptide component showed an obvious upward trend, slowly increased between 50% and 80%, and basically flattened between 80% and 90%, so the preferred extraction solvent was 80%.

[0155] 3.6 Optimization of chromatographic conditions

[0156] 3.6.1 Choice of mobile phase

[0157] The effects of different mobile phase systems (methanol-water, acetonitrile-water, acetonitrile-0.1% phosphoric acid aqueous solution, acetonitrile-0.1% formic acid aqueous solution, acetonitrile-20 mM ammonium acetate aqueous solution) on the separation efficiency of the various cyclic peptide components of Radix Pseudostellariae were investigated. Figure 6 ) as the mobile phase, the chromatographic peaks are difficult to separate by baseline; using acetonitrile-water ( Figure 2 )、Acetonitrile-0.1% phosphoric acid aqueous solution( Figure 7 )、Acetonitrile-0.1% formic acid aqueous solution( Figure 8 )、Acetonitrile-20mmol ammonium acetate aqueous solution ( Fig. 9 ) was used as the mobile phase, the peak shapes of the chromatographic peaks were good and baseline separation could be achieved, so the acetonitrile-water system was finally selected as the preferred mobile phase.

[0158] 3.6.2 Selection of detection wavelength

[0159] The cyclic peptide component absorbs at the terminal end, so 203 nm is the preferred detection wavelength.

[0160] 3.6.3 Selection of relative correction factor calculation method

[0161] There are two methods for calculating the relative correction factor of the measured component in the one-measurement-multiple-evaluation method, namely the slope method and the multi-point correction method. The present invention compares the relative correction factors of each component calculated by the two methods, and shows that there is no significant difference between the two calculation methods. However, considering that the intercept caused by the systematic error and instrument operation error in the standard curve based on the slope method affects the accuracy of the result, the present invention uses the multi-point correction method as the preferred calculation method.

[0162] The present invention establishes a method for simultaneously determining the contents of 7 cyclic peptide components in Radix Pseudostellariae by a one-test-multiple-evaluation method, takes Radix Pseudostellariae cyclic peptide B as an internal reference, and calculates the relative correction factors of Radix Pseudostellariae cyclic peptide A, Radix Pseudostellariae cyclic peptide A, Radix Pseudostellariae cyclic peptide B, Radix Pseudostellariae cyclic peptide D, Radix Pseudostellariae cyclic peptide E and Radix Pseudostellariae cyclic peptide H in Radix Pseudostellariae. And the relative correction factors of the 6 cyclic peptide components are investigated for durability from aspects such as different chromatographic systems, chromatographic columns, column temperatures, and flow rates, and the results show that the method has good durability. There is no significant difference between the calculated values ​​of the one-test-multiple-evaluation method and the measured values ​​of the external standard method for 10 batches of Radix Pseudostellariae samples. The relative correction factor established by the present invention is accurate and reliable, and the one-test-multiple-evaluation method using Radix Pseudostellariae cyclic peptide B as an internal reference effectively fills the gap of Radix Pseudostellariae without content determination index components, provides a new method for Radix Pseudostellariae quality control and evaluation, and improves the quality control level of Radix Pseudostellariae, and the method is simple, accurate, and easy to further promote.

[0163] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for determining the contents of 7 cyclic peptide components in Radix Pseudostellariae by a one-test-multiple-evaluation method, characterized in that: The steps include: (1) Preparation of reference solution: Take 7 kinds of cyclopeptide component reference substances, namely, Radix Pseudostellariae Cyclic Peptide A, Radix Pseudostellariae Cyclic Peptide B ... D, Radix Pseudostellariae Cyclic Peptide E and Radix Pseudostellariae Cyclic Peptide H, add methanol to dissolve them, and obtain reference solution; (2) Preparation of the test solution: Take the Pseudostellariae Radix to be tested sample, add methanol solution for ultrasonic extraction, filter, take the filtrate, add methanol solution to the residue obtained after drying, dissolve it, filter, take the filtrate to obtain the test solution; the ratio of the Pseudostellariae Radix to be tested sample to the methanol solution used for dissolution is 2-5 g: 5-10 mL; (3) Inject the reference solution and the test solution into the HPLC, use Pseudostigma cyclotide B as the internal reference, and calculate the relative correction factor f s / i Calculate the contents of the other 6 cyclic peptide components to be tested; The mobile phase used in the high performance liquid chromatograph in step (3) is as follows: phase A is acetonitrile; phase B is water; The elution method adopted by the high performance liquid chromatograph in step (3) is gradient elution, which is as follows: 0-5min, 20%A; 5-15min, 20%→40%A; 15-30min, 40%A; 30-36min, 40%→80%A; 36-55min, 80%A; 55-56min, 80%→20%A; The relative correction factor f in step (3) s / i The multi-point correction method is used for calculation, and the calculation method is as follows: Relative correction factor f s / i =f s / f i =(C i ×A s ) / (C s ×A i ); Among them, f i is the correction factor for the cyclic peptide component to be tested in the reference solution, C i is the content of the cyclic peptide component to be tested in the reference solution, A i is the peak area of ​​the cyclic peptide component to be tested in the reference solution, f s is the internal reference correction factor in the reference solution, C s is the content of internal reference substance in the reference solution, A s is the peak area of ​​the internal reference substance in the reference solution.

2. The method according to claim 1, characterized in that The concentrations of the seven cyclic peptide component reference substances in the reference substance solution in step (1) are all 10 μg / mL-300 μg / mL.

3. The method according to claim 1, characterized in that Step (2) also includes at least one of the following conditions: ii1) the volume percentage concentration of the methanol solution used in the ultrasonic extraction is 60%-100%; ii2) The ultrasonic extraction time is 0.5h-1.5h.

4. The method according to claim 1, characterized in that: The test conditions of the high performance liquid chromatograph in step (3) include: Chromatographic column: Select a chromatographic column with a packing size of 5 μm; Flow rate: 0.8-1.2mL / min; Column temperature: 25-35°C; Injection volume: 2-25 μL; Detection wavelength: 203nm-220nm.

5. The method according to claim 1, characterized in that The test conditions of the high performance liquid chromatograph in step (3) include: Chromatographic column: YMC Triart C 18 ; Flow rate: 0.8-1.2mL / min; Column temperature: 25-35°C; Injection volume: 5-25 μL; Detection wavelength: 203-220nm.

6. The method according to claim 1, characterized in that The relative retention times of Pseudostellariae Radix A, Pseudostellariae Radix A, Pseudostellariae Radix B, Pseudostellariae Radix D, Pseudostellariae Radix E and Pseudostellariae Radix H relative to Pseudostellariae Radix B are 1.07-1.38, 0.61-0.87, 0.70-0.99, 0.87-1.23, 1.25-1.74 and 1.14-1.53 ​​respectively.

7. The method according to claim 1, characterized in that The relative correction factor f of Pseudoradix ... s / i They are 1.11-1.62, 0.82-1.20, 0.73-1.18, 0.78-1.34, 0.81-1.35 and 0.58-1.25 respectively.

8. The method according to claim 1, characterized in that The content of the cyclic peptide component to be tested in step (3) is calculated as follows: K x =f s / i ×(K s ×T x / T s ); Among them, T x and T s are the peak areas of the cyclic peptide component to be tested and the internal reference in the test solution, respectively. x and K s are the contents of the cyclic peptide component to be tested and the internal reference substance in the test solution, respectively. s / i is the relative correction factor of the cyclic peptide component to be measured.

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