Nuclear magnetic resonance identification method of sea sodium polysaccharide

Abstract

The invention relates to the technical field of medicines, and provides a nuclear magnetic resonance identification method of sea sodium polysaccharide. The method comprises the following steps: mixing a to-be-detected sample, an internal standard substance and heavy water to obtain a to-be-detected solution, then carrying out nuclear magnetic resonance detection on the to-be-detected solution, and analyzing the obtained nuclear magnetic resonance hydrogen spectrum to identify whether the to-be-detected sample is a sodium polysaccharide qualified product or not, the nuclear magnetic resonance detection conditions comprise that the frequency is not less than 600MHz, the pulse angle is 90 degrees, the acquisition time is not less than 2s, the relaxation time is not less than 12s, the scanning times are not less than 16s, and the spectral width at least comprises a range of 10ppm to 0ppm. According to the method, the nuclear magnetic resonance spectroscopy is adopted for the first time to establish the identification method of the sea sodium polysaccharide, operation is easy, specificity is high, and effective identification of the sea sodium polysaccharide can be achieved.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to a nuclear magnetic resonance identification method for sodium thunbergii polysaccharide. Background Art

[0002] The body wall of sea cucumber contains abundant polysaccharides, mainly including two categories: fucosylated chondroitin sulfate and fucosylated chondroitin sulfate. Among them, sea cucumber fucosylated chondroitin sulfate has rich biological activities due to its unique structure, such as lowering blood lipids, anti-tumor, anti-virus, anti-inflammatory, etc., especially good anticoagulant and anti-thrombotic activities.

[0003] At present, there are few pharmacopoeia identification methods for polysaccharide drugs, which generally include total sugar content determination, monosaccharide composition analysis by thin layer chromatography or infrared identification, but the specificity of these methods is poor. At present, there is no identification method suitable for sodium thunbergii polysaccharide in the art. Summary of the invention

[0004] In view of this, the present invention provides a nuclear magnetic resonance identification method for sodium thunbergii polysaccharide. The identification method provided by the present invention is simple to operate, has strong specificity, and can achieve effective identification of sodium thunbergii polysaccharide.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] A nuclear magnetic resonance identification method for sodium thunbergii polysaccharide comprises the following steps:

[0007] Mixing the sample to be tested, the internal standard and heavy water to obtain a test solution;

[0008] Performing nuclear magnetic resonance detection on the test liquid to obtain a nuclear magnetic resonance hydrogen spectrum;

[0009] Analyzing the nuclear magnetic resonance hydrogen spectrum, and identifying whether the sample to be tested is a qualified product of sodium thunbergii polysaccharide according to the analysis result;

[0010] The conditions of the nuclear magnetic resonance detection include: a frequency of not less than 600 MHz, a pulse angle of 90°, an acquisition time of not less than 2 seconds, a relaxation time of not less than 12 seconds, a scanning number of not less than 16 times, and a spectrum width of at least 10 ppm to 0 ppm;

[0011] The nuclear magnetic resonance hydrogen spectrum standards of the qualified product of sodium hyaluronate polysaccharide include:

[0012] The nuclear magnetic resonance hydrogen spectrum contains signal peaks of fucose terminal hydrogen, N-acetyl methyl hydrogen and fucose methyl hydrogen; relative to the methyl signal of the internal standard, the relative displacement of the fucose terminal hydrogen signal peak is 5.68±0.03ppm, the relative displacement of the N-acetyl methyl hydrogen signal peak is 2.08±0.03ppm, and the fucose methyl hydrogen signal peak is a double peak centered at a relative displacement of 1.38±0.03ppm;

[0013] The signal-to-noise ratio of the N-acetylmethyl hydrogen signal peak in the H NMR spectrum is above 2000 / 1 in the region close to the relative displacement of 2ppm;

[0014] In the relative shift range of 0.2-0.8ppm and 6.5-10ppm of the nuclear magnetic resonance hydrogen spectrum, there is no unidentified signal greater than 8% of the signal peak intensity of fucose methyl hydrogen; in the relative shift range of 3-6ppm of the nuclear magnetic resonance hydrogen spectrum, there is no unidentified signal greater than 120% of the signal peak intensity of fucose methyl hydrogen; the intensity of the fucose methyl hydrogen signal peak is measured as the average intensity of the double peak centered at the relative shift of 1.38±0.03ppm;

[0015] The integral area ratio of the N-acetyl methyl hydrogen signal peak and the fucose methyl hydrogen signal peak in the nuclear magnetic resonance hydrogen spectrum is 1.2 to 1.8; the relative displacement integral range of the N-acetyl methyl hydrogen signal peak is 1.8 to 2.4 ppm, and the relative displacement integral range of the fucose methyl hydrogen signal peak is 1.1 to 1.6 ppm;

[0016] The qualified product of the sodium hyaluronate polysaccharide is a fucosylated chondroitin sulfate polysaccharide; the weight average molecular weight of the sodium hyaluronate polysaccharide is 90,000 to 130,000, the monosaccharide composition includes glucuronic acid, N-acetylgalactose and fucose, the molar ratio of glucuronic acid, N-acetylgalactose and fucose is 1:0.8 to 1.2:0.5 to 0.8; the mass percentage of sulfate groups in the sodium hyaluronate polysaccharide is 25 to 40%.

[0017] Preferably, the concentration of the sample to be tested in the test solution is above 20 mg / mL.

[0018] Preferably, the concentration of the sample to be tested in the test solution is 20-30 mg / mL.

[0019] Preferably, the internal standard is sodium 3-(trimethylsilyl)deuterated propionate.

[0020] Preferably, the concentration of the internal standard in the test solution is 0.002% (w / v).

[0021] Preferably, the acquisition time of the nuclear magnetic resonance detection is 2 to 3 seconds.

[0022] Preferably, the relaxation time of the nuclear magnetic resonance detection is 12 to 16 seconds.

[0023] Preferably, the number of scans of the nuclear magnetic resonance detection is 16 to 32 times.

[0024] Preferably, the instrument mode of the nuclear magnetic resonance detection is a pulse Fourier transform mode.

[0025] Preferably, the temperature of the nuclear magnetic resonance detection is 20-30°C.

[0026] The present invention provides a method for identifying sodium thunbergii polysaccharide by nuclear magnetic resonance. The method comprises mixing a sample to be tested, an internal standard and heavy water to obtain a liquid to be tested, and then performing nuclear magnetic resonance detection on the liquid to be tested, and analyzing the obtained nuclear magnetic resonance hydrogen spectrum to identify whether the sample to be tested is a qualified product of sodium thunbergii polysaccharide. Sodium thunbergii polysaccharide is a natural fucosylated chondroitin sulfate polysaccharide extracted by the inventor from the body wall of sea cucumbers, which can target the terminal rate-limiting enzyme iFXase in the endogenous coagulation pathway, has good anticoagulant effect, and has little bleeding side effect, thereby achieving the effect of anticoagulation and no bleeding. The present invention adopts nuclear magnetic resonance spectroscopy to establish an identification method of sodium thunbergii polysaccharide for the first time, which is simple to operate, has strong specificity, and can achieve effective identification of sodium thunbergii polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Sodium hyaluronate 1 HNMR test spectrum;

[0028] Figure 2 Sodium hyaluronate 13 CNMR test spectrum;

[0029] Figure 3 This is the HSQC test spectrum of sodium thunbergii polysaccharide;

[0030] Figure 4 For the standard solution of sample number 400-30-2-2-8 1 H NMR spectrum;

[0031] Figure 5 For the standard solution of sample number 600-30-2-2-8 1 H NMR spectrum;

[0032] Figure 6 For the standard solution of sample number 600-90-2-2-8 1 H NMR spectrum;

[0033] Figure 7 For the standard solution of sample number 600-90-0.8-2-8 1 HNMR spectrum;

[0034] Figure 8 For the standard solution of sample number 600-90-3-2-8 1 H NMR spectrum;

[0035] Fig. 9 For the standard solution of sample number 600-90-2-4-8 1 H NMR spectrum;

[0036] Fig.10 For the standard solution of sample number 600-90-2-8-8 1 H NMR spectrum;

[0037] Fig.11 For the standard solution of sample number 600-90-2-12-8 1 H NMR spectrum;

[0038] Fig.12 For the standard solution of sample number 600-90-2-16-8 1 H NMR spectrum;

[0039] Fig.13 For the standard solution of sample number 600-90-2-12-4 1 H NMR spectrum;

[0040] Fig.14 For the standard solution of sample number 600-90-2-12-8 1 H NMR spectrum;

[0041] Fig.15 For the standard solution of sample number 600-90-2-12-16 1 H NMR spectrum;

[0042] Fig.16 For the standard solution of sample number 600-90-2-12-32 1 H NMR spectrum;

[0043] Fig.17 For the standard solution of sample number 600-90-2-12-16-5 1 H NMR spectrum;

[0044] Fig.18 For the standard solution of sample number 600-90-2-12-16-10 1 H NMR spectrum;

[0045] Fig.19 For the standard solution of sample number 600-90-2-12-16-20 1H NMR spectrum;

[0046] Fig. 20 For the standard solution of sample number 600-90-2-12-16-30 1 H NMR spectrum;

[0047] Fig.21 For the system suitability solution with sample number 400-30-2-2-8 1 H NMR spectrum;

[0048] Fig. 22 For the system suitability solution with sample number 600-30-2-2-8 1 H NMR spectrum;

[0049] Fig.23 For the system suitability solution with sample number 600-90-2-2-8 1 H NMR spectrum;

[0050] Fig.24 For the system suitability solution of sample number 600-90-0.8-2-8 1 HNMR spectrum;

[0051] Fig.25 For the system suitability solution of sample number 600-90-3-2-8 1 H NMR spectrum;

[0052] Fig.26 For the system suitability solution of sample number 600-90-2-4-8 1 H NMR spectrum;

[0053] Fig. 27 For the system suitability solution with sample number 600-90-2-8-8 1 H NMR spectrum;

[0054] Fig.28 For the system suitability solution of sample number 600-90-2-12-8 1 H NMR spectrum;

[0055] Fig.29 For the system suitability solution of sample number 600-90-2-16-8 1 H NMR spectrum;

[0056] Fig.30 For the system suitability solution of sample number 600-90-2-12-4 1 H NMR spectrum;

[0057] Fig.31 For the system suitability solution with sample number 600-90-2-12-16 1 H NMR spectrum;

[0058] Fig.32 For the system suitability solution of sample number 600-90-2-12-32 1 H NMR spectrum.

[0059] Fig.33 The first test result of the system suitability solution in Example 3 1 HNMR spectrum;

[0060] Fig.34 The second test result of the system suitability solution in Example 3 1 HNMR spectrum;

[0061] Fig.35 The third test result of the system suitability solution in Example 3 1 HNMR spectrum;

[0062] Fig.36 The fourth test result of the system suitability solution in Example 3 1 HNMR spectrum;

[0063] Fig.37 The results of the fifth test of the system suitability solution in Example 3 1 HNMR spectrum;

[0064] Fig.38 The 202405 batches of sodium hyaluronate polysaccharide in Example 3 1 HNMR spectrum;

[0065] Fig.39 The 202404 batches of sodium hyaluronate polysaccharide in Example 3 1 HNMR spectrum;

[0066] Fig.40 The 202403 batches of sodium hyaluronate polysaccharide in Example 3 1 HNMR spectrum;

[0067] Fig.41 The 202402 batches of sodium hyaluronate polysaccharide in Example 3 1 HNMR spectrum;

[0068] Fig.42 The 202401 batches of sodium hyaluronate polysaccharide in Example 3 1 HNMR spectrum. DETAILED DESCRIPTION

[0069] First, sodium thunbergii polysaccharide will be described.

[0070] In the present invention, the sea sodium polysaccharide refers to a natural polysaccharide extracted from the body wall of a sea cucumber, and the sea cucumber includes but is not limited to one or more of the sea cucumbers of the jade foot, rough sea cucumber, sea cucumber, black sea cucumber and black milk sea cucumber, preferably the sea cucumber of the jade foot (Latin name: Holothurialeucospilota (Brandt)); the sea sodium polysaccharide is a fucosylated chondroitin sulfate polysaccharide; the weight average molecular weight of the sea sodium polysaccharide is 90,000 to 130,000, the polydispersity index is ≤2, the monosaccharide composition includes glucuronic acid, N-acetylgalactose and fucose, and the molar ratio of glucuronic acid, N-acetylgalactose and fucose is 1:0.8 to 1.2:0.5 to 0.8; the mass percentage of sulfate groups in the sea sodium polysaccharide is 25 to 40%.

[0071] The structural unit of sodium thunbergii polysaccharide is shown in Formula I:

[0072]

[0073] In formula I: R1 is H, SO3 - or fucosyl, R2 is H or SO3 - , R3 is H or SO3 - , R4 is H or fucosyl; at least one of R1 and R4 is fucosyl; X ⊕ represents a cation;

[0074] The structure of the fucosyl in R1 and R4 is shown in Formula I-1:

[0075]

[0076] In formula I-1: R5 is H or SO3 - , R6 is H or SO3 - , R7 is H or SO3 - .

[0077] The cation in formula I is a hydrogen ion, an alkali metal ion, an alkaline earth metal ion, an ammonium ion or an iron ion, specifically a hydrogen ion, a potassium ion, a sodium ion, a calcium ion, an ammonium ion or an iron ion, preferably a sodium ion; when the cation is a sodium ion, the mass percentage of the sodium ion in the sodium ion polysaccharide is 5 to 15%.

[0078] The results of the evaluation of the anticoagulant activity of sodium hyaluronate polysaccharide on mice showed that sodium hyaluronate polysaccharide can significantly prolong the APTT coagulation time of mice and rats, without affecting the PT and TT coagulation times. The above research results indicate that sodium hyaluronate polysaccharide targets the intrinsic coagulation pathway and does not affect the extrinsic coagulation pathway.

[0079] Referring to the heparin bioassay method in General Chapter 1208 of the Pharmacopoeia, the activity of sodium hyaluronate polysaccharide against factor IIa, factor Xa, ATⅢ-Ⅱa, and ATⅢ-Xa was tested. The results showed that at an experimental dose of 500 μg / mL, sodium hyaluronate polysaccharide showed no activity against factor IIa, factor Xa, and ATⅢ-Ⅱa, and only had a weak effect on ATⅢ-Xa (inhibition rate of 36.7%). The activity of the rate-limiting enzyme of the endogenous coagulation pathway, "intrinsic factor coagulation factor X enzyme (iFXase)", was tested by ELISA. The results showed that the test drug could significantly inhibit the rate-limiting enzyme iFXase of the endogenous coagulation pathway, IC 50 The value is 207.5ng / mL; the above experiment further clarified from the mechanism that sodium thiazolinone polysaccharide can selectively inhibit the activity of iFXase, the rate-limiting enzyme in the intrinsic coagulation pathway, and target the intrinsic coagulation pathway.

[0080] The identification method of the present invention is described in detail below.

[0081] The present invention provides a nuclear magnetic resonance identification method for sodium thunbergii polysaccharide, comprising the following steps:

[0082] Mixing the sample to be tested, the internal standard and heavy water to obtain a test solution;

[0083] Performing nuclear magnetic resonance detection on the test liquid to obtain a nuclear magnetic resonance hydrogen spectrum;

[0084] The nuclear magnetic resonance hydrogen spectrum is analyzed, and whether the sample to be tested is a qualified product of sodium thunbergii polysaccharide is identified according to the analysis result.

[0085] The present invention mixes a sample to be tested, an internal standard and heavy water to obtain a test solution. In the present invention, the internal standard is preferably 3-(trimethylsilyl) sodium deuterated propionate; the concentration of the sample to be tested in the test solution is preferably 20 mg / mL or more, preferably 20 to 30 mg / mL, and the concentration of the internal standard in the test solution is preferably 0.002% (w / v).

[0086] After obtaining the test liquid, the present invention performs nuclear magnetic resonance detection on the test liquid to obtain a nuclear magnetic resonance hydrogen spectrum. In the present invention, the conditions of the nuclear magnetic resonance detection preferably include: a frequency of not less than 600 MHz, a pulse angle of 90°, an acquisition time of not less than 2 seconds, preferably 2 to 3 seconds, a relaxation time of not less than 12 seconds, preferably 12 to 16 seconds, a scanning number of not less than 16 times, preferably 16 to 32 times, and a spectrum width of at least 10 ppm to 0 ppm.

[0087] In the present invention, the instrument mode of the nuclear magnetic resonance detection is preferably a pulse Fourier transform mode; the temperature of the nuclear magnetic resonance detection is preferably 20-30°C. In a specific embodiment of the present invention, the nuclear magnetic resonance detection is preferably performed at room temperature.

[0088] After obtaining the nuclear magnetic resonance hydrogen spectrum, the present invention analyzes the nuclear magnetic resonance hydrogen spectrum and identifies whether the sample to be tested is a qualified product of sodium thunbergii polysaccharide according to the analysis result. In the present invention, the nuclear magnetic resonance hydrogen spectrum standard of the qualified product of sodium thunbergii polysaccharide includes:

[0089] In the nuclear magnetic resonance hydrogen spectrum, there are signal peaks of fucose terminal hydrogen (recorded as signal 1), N-acetyl methyl hydrogen (recorded as signal 2) and fucose methyl hydrogen (recorded as signal 3); relative to the methyl signal of the internal standard (i.e., sodium 3-(trimethylsilyl)deuterated propionate), the relative displacement of the fucose terminal hydrogen signal peak is 5.68±0.03ppm, the relative displacement of the N-acetyl methyl hydrogen signal peak is 2.08±0.03ppm, and the fucose methyl hydrogen signal peak is a double peak centered at a relative displacement of 1.38±0.03ppm;

[0090] The signal-to-noise ratio of the N-acetylmethyl hydrogen signal peak in the nuclear magnetic resonance hydrogen spectrum is above 2000 / 1;

[0091] In the relative shift range of 0.2-0.8ppm and 6.50-10ppm of the nuclear magnetic resonance hydrogen spectrum, there is no unidentified signal greater than 8% of the signal peak intensity of fucose methyl hydrogen; in the relative shift range of 3-6ppm of the nuclear magnetic resonance hydrogen spectrum, there is no unidentified signal greater than 120% of the signal peak intensity of fucose methyl hydrogen; the intensity of the fucose methyl hydrogen signal peak is measured as the average intensity of the double peak centered at the relative shift of 1.38±0.03ppm;

[0092] The integral area ratio of the N-acetyl methyl hydrogen signal peak and the fucose methyl hydrogen signal peak in the nuclear magnetic resonance hydrogen spectrum is 1.2-1.8; the relative displacement integral range of the N-acetyl methyl hydrogen signal peak is 1.8-2.4 ppm, and the relative displacement integral range of the fucose methyl hydrogen signal peak is 1.1-1.6 ppm.

[0093] In the present invention, the displacements in the H NMR spectrum are all relative displacements relative to the methyl signal peak of 3-(trimethylsilyl) sodium deuterated propionate, wherein the methyl signal peak of 3-(trimethylsilyl) sodium deuterated propionate is 0.00 ppm, which will not be described in detail later.

[0094] In the present invention, if the nuclear magnetic resonance hydrogen spectrum of the sample to be tested meets the above standards, the sample to be tested can be judged as a qualified product of sodium thunbergii polysaccharide, otherwise it is an unqualified product.

[0095] The sodium sea polysaccharide of the present invention is fucosylated chondroitin sulfate, and the skeleton is a copolymer of disaccharide units composed of acetylgalactosamine and glucuronic acid, and the structural difference is only the difference in the content of fucosyl and sulfate ester groups and the modification site. According to the nuclear magnetic resonance hydrogen spectrum, the present invention determines that the characteristic hydrogen signal in the sodium sea polysaccharide structure is fucose terminal hydrogen (5.0-6.0ppm), wherein 5.68±0.03ppm can be obviously distinguished from other signal peaks, and the acetylmethyl hydrogen 2.08±0.03ppm and fucose methyl hydrogen 1.38±0.03ppm in acetylgalactosamine are also obviously distinguished from other signal peaks. Therefore, the present invention selects fucose terminal hydrogen, N-acetylmethyl hydrogen and fucose methyl hydrogen as characteristic peaks for identifying sodium sea polysaccharide.

[0096] In addition, signals generated by sodium thunbergii polysaccharide and signals of heavy water are observed between 2.08ppm and 5.00ppm in the hydrogen nuclear magnetic resonance spectrum.

[0097] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0098] Example 1 Extraction of sodium thunbergii polysaccharide

[0099] After the body wall of Holothuria leucospilota (Brandt) was carefully separated from other tissues, it was immediately immersed in acetone and stored at 4°C for 24 hours. The dried tissue (500 g) was ground into powder, suspended in 10 L of 0.1 mol / L sodium acetate buffer (pH 6) containing 50 g of papain, incubated at 65°C for 24 hours, and then the incubation solution was centrifuged at low temperature (4000 rpm, 4°C for 30 minutes) to obtain the enzymatic supernatant. 6 mol / L hydrochloric acid aqueous solution was added to the enzymatic supernatant to adjust the pH to 2.5±0.5, reacted for 2 hours under stirring, and centrifuged at low temperature (4000 rpm, 4°C for 30 minutes) to obtain the acid hydrolysis supernatant. Add 40wt% sodium hydroxide aqueous solution to the acid hydrolysis supernatant to adjust the pH value to 7.0±0.5. Under stirring, add 95vol% ethanol in a volume of 1 times the supernatant to the reaction system. After maintaining at -10°C for 24h, centrifuge at low temperature (4000rpm, centrifuge at 4°C for 30min) to collect the formed precipitate. The precipitate is dissolved in 500mL of distilled water, and 1L95vol% ethanol is added. After maintaining at -10°C for 24h, centrifuge at low temperature (4000rpm, centrifuge at 4°C for 30min) to collect the formed precipitate. Add 500mL95vol% ethanol to the precipitate for washing, centrifuge at low temperature (4000rpm, centrifuge at 4°C for 30min), collect the solid, dissolve in 500mL of distilled water, and freeze-dry to obtain 4g of crude sea sodium polysaccharide.

[0100] 4g of crude sodium polysaccharide was placed on a DEAE cellulose column (10cm×4cm) pre-equilibrated with 0.1mol / L HAc-NaAc buffer (pH 6), and washed with 5L of HAc-NaAc buffer (pH 6) containing 0.5mol / L NaCl and 2L of HAc-NaAc buffer (pH 6) containing 1mol / L NaCl, respectively, with a column flow rate of 50mL / min, and 500mL of each fraction was collected. Each fraction was detected by molecular weight and molecular weight distribution HPLC method, and the chromatographic peak fraction with a weight average molecular weight of 90,000 to 130,000 was collected, and 1 volume of 95vol% ethanol of the effluent was added, and after maintaining at -10°C for 24h, low-temperature centrifugation (4000rpm, 4°C centrifugation for 30min) was performed to collect the formed precipitate. The precipitate was dissolved in distilled water and concentrated to 1 / 2 of the original volume using an ultrafiltration membrane with a molecular weight cutoff of 10,000, water was added to the original volume, ultrafiltration was performed to 1 / 2 of the volume, water was added to the original volume, and then concentrated to 1 / 2 of the original volume, water was added to the original volume, and then concentrated to 1 / 2 of the original volume, and then water was added to the original volume, and then concentrated to 1 / 2 of the original volume, and the concentrate was collected and freeze-dried to obtain 2.5 g of pure sodium polysaccharide (the cation is sodium). According to the above operation, 5 batches of parallel samples were continuously prepared, namely 202401, 202402, 202403, 202404 and 202405 batches of sodium polysaccharide.

[0101] Example 2 Establishment of the Nuclear Magnetic Resonance Identification Method for Sodium Hyaluronate Polysaccharide

[0102] 1. Confirmation of exclusivity

[0103] Determination of sodium hyaluronate (batch number: 202401) 1 H. 13 C. HSQC NMR spectra (such as Figure 1 to Figure 3 As shown), confirm the attribution of the characteristic peaks of the standard, and establish the specificity standard of the identification method. The sodium sea polysaccharide of the present invention is a fucosylated chondroitin sulfate, and the skeleton is a copolymer of disaccharide units composed of acetylgalactosamine and glucuronic acid, and the structural difference is only the difference in the content of fucosyl and sulfate ester groups and the modification site. According to the two-dimensional nuclear magnetic resonance spectrum, it is determined that the characteristic hydrogen signal in the structure of sodium sea polysaccharide is fucose terminal hydrogen (5.0-6.0ppm), of which 5.68±0.03ppm can be clearly distinguished from other signal peaks; acetylmethyl hydrogen 2.08±0.03ppm and fucose methyl hydrogen 1.38±0.03ppm in acetylgalactosamine are also clearly distinguished from other signal peaks. Therefore, fucose terminal hydrogen, N-acetylmethyl hydrogen and fucose methyl hydrogen are selected as characteristic peaks for identifying sodium sea polysaccharide, wherein fucose methyl hydrogen is a double peak centered at 1.38±0.03ppm.

[0104] 2. Solution Preparation

[0105] Standard solution: Dissolve the identification standard of Sodium Hyaluronate (Batch No.: 202401) in heavy water to prepare a solution of a certain concentration, and add 0.002% (w / v) of 3-(trimethylsilyl) sodium deuterated propionate (TSP) as an internal standard.

[0106] System suitability solution: Dissolve the oversulfated chondroitin sulfate reference substance (Oversulfated Chondroitin Sulfate RS) in the standard solution to prepare a solution with a concentration of 0.3% (w / w).

[0107] The sources of the reagents used to prepare the solution are as follows: heavy water (99.9%, Shanghai Bid Pharmaceutical Technology Co., Ltd.); sodium 3-(trimethylsilyl)deuterated propionate (98 atomic % D, Beijing Bailingwei Technology Co., Ltd.); polysulfated chondroitin sulfate (batch number: 140789-202202, China Food and Drug Inspection Institute).

[0108] 3. NMR instrument test condition screening

[0109] Instrument: Bruker 600MHz AVANCENEO (No.H03128OB / 05.02 / R / 6852)

[0110] Bruker400 MHzAVANCE III HD (No.2138276 / ECL02.00 / 00311).

[0111] Software: Topspin4.3.1

[0112] The instrument frequency, test pulse angle, acquisition time, relaxation time, scan number and sample concentration were examined using standard solutions and system suitability solutions. The signal-to-noise ratio of the N-acetyl methyl hydrogen signal peak (abbreviated as acetyl signal-to-noise ratio) and the double-peak signal intensity of fucose methyl hydrogen (recorded as methyl peak 1 and methyl peak 2) were used as the inspection standards. The test results are shown in Tables 1 to 2 and attached. Figures 4 to 32 .

[0113] Table 1 Test results of standard solution conditions

[0114]

[0115] In Table 1, the numbers in the sample number represent the frequency, pulse angle, acquisition time, relaxation time and number of scans, for example, 400-30-2-2-8 means that the test is conducted under the conditions of a frequency of 400 MHz, a pulse angle of 30°, an acquisition time of 2 seconds, a relaxation time of 2 seconds and a scan number of 8 times. The concentration of the standard in the standard solution used in the frequency, pulse angle, acquisition time, relaxation time and scan number tests is 20 mg / mL, and the last digit of the sample number in the sample concentration test represents the sample concentration value.

[0116] Table 2 System suitability solution condition examination test results

[0117]

[0118] In Table 2, the numbers in the sample number represent the frequency, pulse angle, acquisition time, relaxation time and number of scans respectively. For example, 400-30-2-2-8 means that the test is carried out under the conditions of a frequency of 400 MHz, a pulse angle of 30°, an acquisition time of 2 seconds, a relaxation time of 2 seconds and a scan number of 8 times; in the examination of system suitability solution conditions, the concentration of the standard in the system suitability solution used is 20 mg / mL.

[0119] Through the above test results, according to the signal-to-noise ratio, characteristic peak intensity and chemical shift results, and comprehensive practical evaluation such as sample dosage and test time, the nuclear magnetic resonance test conditions are determined as follows: instrument mode: pulse Fourier transform; frequency: not less than 600MHz (for 1H); temperature: 20℃~30℃; pulse angle: 90°; acquisition time: not less than 2 seconds; relaxation time: at least 12 seconds; number of scans: not less than 16 times, spectral width: at least 10~0ppm.

[0120] The NMR test conditions used in subsequent experiments were: instrument mode: pulse Fourier transform; frequency: 600 MHz; pulse angle: 90°; acquisition time: 2 seconds; relaxation time: 12 seconds; scan number: 16 times. The test temperature was room temperature.

[0121] According to the established test parameters, the standard solution was used for testing, and the mean value of the double peak signal intensity of fucose methyl hydrogen in the standard NMR hydrogen spectrum (recorded as the mean value of the methyl peak intensity), the integral area of ​​the N-acetyl methyl hydrogen signal peak (signal 2) and the fucose methyl hydrogen signal peak (signal 3) were tested, and the ratio (recorded as acetyl integral / methyl integral) was calculated, wherein the integral range of signal 2 was 1.80-2.40ppm; the integral range of signal 3 was 1.10-1.60ppm. The results are shown in Table 3.

[0122] Table 3 Standard solution test results

[0123]

[0124] The peak intensity in Table 3 refers to the intensity of the strongest signal peak present within 0.2-0.8 ppm, 3.0-6.0 ppm or 6.50-10 ppm.

[0125] 4. Applicability requirements

[0126] Based on the above experimental results, the applicability requirements of the NMR identification method of sodium thunbergii polysaccharide were determined:

[0127] 1) Number of scans: Adjust the number of scans until the signal-to-noise ratio of the N-acetylmethyl hydrogen signal of sodium hyaluronate in the standard solution reaches at least 2000 / 1.

[0128] 2) Chemical shift: The TSP (sodium 3-(trimethylsilyl)deuterated propionate) methyl signal of all samples was set to 0.00 ppm; the N-acetyl methyl hydrogen resonance of sodium thiamethoxam and persulfated chondroitin sulfate in the system suitability solution should be observed at 2.08±0.03 ppm and 2.14±0.03 ppm, respectively.

[0129] 3) Draw a baseline from 10.00ppm to 0.00ppm. The displacement values ​​of the terminal hydrogen of fucose (signal 1), N-acetyl methyl hydrogen of acetylgalactosamine (signal 2), and methyl hydrogen of fucose (signal 3) in sodium thiocyanate are located at 5.68±0.03ppm, 2.08±0.03ppm, and 1.38±0.03ppm (a double peak centered at 1.38±0.03ppmppm), respectively. Between 2.08ppm (signal 2) and 5.00ppm, the signal generated by sodium thiocyanate and the signal of D2O (heavy water, used as a solvent, will appear in the NMR spectrum) will be seen.

[0130] 4) Acceptance limit: In the range of 0.20-0.80ppm and 6.50-10.00ppm, there is no unidentified signal greater than 8% of the average intensity of the double peak of signal 3; in the range of 3.00-6.00ppm, there is no unidentified signal greater than 120% of the average intensity of the double peak of signal 3. The integral area ratio of signal 2 to signal 3 is 1.2-1.8, of which the integral range of signal 2 is 1.8-2.4ppm, and the integral range of signal 3 is 1.1-1.6ppm.

[0131] Example 3 Sample determination

[0132] 1. Solution Preparation

[0133] Standard solution: Dissolve the sodium thiamethoxam polysaccharide identification standard (batch number: 202401) in heavy water to prepare a solution with a concentration of 20 mg / mL, and add 0.002% (w / v) of 3-(trimethylsilyl) sodium deuterated propionate (TSP) as an internal standard.

[0134] System suitability solution: Dissolve the oversulfated chondroitin sulfate reference substance (Oversulfated Chondroitin Sulfate RS) in the standard solution to prepare a solution with a concentration of 0.3% (w / w).

[0135] Sample solution: The sodium thunbergii polysaccharide samples to be identified (batch numbers 202401, 202402, 202403, 202404, and 202405) were dissolved in heavy water to prepare a solution with a concentration of not less than 20 mg / mL, and 0.002% (w / v) TSP was added.

[0136] 2. Determination of NMR spectra

[0137] A 600 MHz nuclear magnetic resonance spectrometer was used with a pulse angle of 90°, an acquisition time of 2 seconds, a relaxation time of 12 seconds, 16 scans, and a pulse Fourier transform instrument mode. The hydrogen nuclear magnetic resonance spectrum was measured at room temperature.

[0138] The suitability of the assay system was tested 5 times in a row, and the signal-to-noise ratio of the acetyl group was greater than 2000, and the RSD of the mean intensity of the fucose methyl peak was 0.026%. The results are shown in Table 4. 1 HNMR spectrum see Figures 33-37 .

[0139] Table 4 System suitability test results

[0140]

[0141] The test results of the sample solution are shown in Table 5.

[0142] Table 5 Test results of each batch of samples

[0143]

[0144] In Table 5: peak intensity refers to the intensity of the strongest signal peak existing in 0.2-0.8ppm, 3.0-6.0ppm or 6.50-10ppm, methyl peak intensity mean refers to the average value of the double peak intensity of fucose methyl proton, end group shift is the shift of fucose end group hydrogen, acetyl shift is the shift of acetyl methyl hydrogen, methyl peak shift refers to the shift of fucose methyl proton (double peak center), acetyl integration range is 1.8-2.4ppm; methyl integration range is 1.1-1.6ppm.

[0145] Different batches of sodium hyaluronate 1 H NMR spectrum is shown in Figures 38 to 42 .

[0146] According to Table 5 and Figures 38 to 42 It can be seen that the present invention can achieve effective identification of sodium thunbergii polysaccharide, and has simple operation and strong specificity.

[0147] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for identifying sodium thunbergii polysaccharide by nuclear magnetic resonance, characterized in that: The following steps are involved: Mixing the sample to be tested, the internal standard and heavy water to obtain a test solution; Performing nuclear magnetic resonance detection on the test liquid to obtain a nuclear magnetic resonance hydrogen spectrum; Analyzing the nuclear magnetic resonance hydrogen spectrum, and identifying whether the sample to be tested is a qualified product of sodium thunbergii polysaccharide according to the analysis result; The conditions of the nuclear magnetic resonance detection include: a frequency of not less than 600 MHz, a pulse angle of 90°, an acquisition time of not less than 2 seconds, a relaxation time of not less than 12 seconds, a scanning number of not less than 16 times, and a spectrum width of at least 10 ppm to 0 ppm; The nuclear magnetic resonance hydrogen spectrum standards of the qualified product of sodium hyaluronate polysaccharide include: The nuclear magnetic resonance hydrogen spectrum contains signal peaks of fucose terminal hydrogen, N-acetyl methyl hydrogen and fucose methyl hydrogen; relative to the methyl signal of the internal standard, the relative displacement of the fucose terminal hydrogen signal peak is 5.68±0.03ppm, the relative displacement of the N-acetyl methyl hydrogen signal peak is 2.08±0.03ppm, and the fucose methyl hydrogen signal peak is a double peak centered at a relative displacement of 1.38±0.03ppm; The signal-to-noise ratio of the N-acetylmethyl hydrogen signal peak in the nuclear magnetic resonance hydrogen spectrum is above 2000 / 1; In the relative displacement range of 0.2-0.8ppm and 6.5-10ppm of the nuclear magnetic resonance hydrogen spectrum, there is no unidentified signal greater than 8% of the signal peak intensity of fucose methyl hydrogen, and in the relative displacement range of 3-6ppm, there is no unidentified signal greater than 120% of the signal peak intensity of fucose methyl hydrogen; the intensity of the fucose methyl hydrogen signal peak is measured as the average intensity of the double peak centered at the relative displacement of 1.38±0.03ppm; The integral area ratio of the N-acetyl methyl hydrogen signal peak and the fucose methyl hydrogen signal peak in the nuclear magnetic resonance hydrogen spectrum is 1.2 to 1.8; the relative displacement integral range of the N-acetyl methyl hydrogen signal peak is 1.8 to 2.4 ppm, and the relative displacement integral range of the fucose methyl hydrogen signal peak is 1.1 to 1.6 ppm; The qualified product of the sodium hyaluronate polysaccharide is a fucosylated chondroitin sulfate polysaccharide; the weight average molecular weight of the sodium hyaluronate polysaccharide is 90,000 to 130,000, the monosaccharide composition includes glucuronic acid, N-acetylgalactose and fucose, the molar ratio of glucuronic acid, N-acetylgalactose and fucose is 1:0.8 to 1.2:0.5 to 0.8; the mass percentage of sulfate groups in the sodium hyaluronate polysaccharide is 25 to 40%.

2. The identification method according to claim 1, characterized in that: The concentration of the sample to be tested in the test solution is above 20 mg / mL.

3. The identification method according to claim 2, characterized in that: The concentration of the sample to be tested in the test solution is 20-30 mg / mL.

4. The identification method according to claim 1 or 2, characterized in that: The internal standard is sodium 3-(trimethylsilyl)deuterated propionate.

5. The identification method according to claim 1 or 2, characterized in that: The concentration of the internal standard in the test solution is 0.002% (w / v).

6. The identification method according to claim 1, characterized in that: The acquisition time of the nuclear magnetic resonance detection is 2 to 3 seconds.

7. The identification method according to claim 1, characterized in that: The relaxation time of the nuclear magnetic resonance detection is 12 to 16 seconds.

8. The identification method according to claim 1, characterized in that: The scanning times of the nuclear magnetic resonance detection are 16 to 32 times.

9. The identification method according to claim 1, characterized in that: The instrument mode of the nuclear magnetic resonance detection is a pulse Fourier transform mode.

10. The identification method according to claim 1, characterized in that: The temperature of the nuclear magnetic resonance detection is 20-30°C.

Images