A method for detecting polysaccharide substances in a Tan Re Qing injection

Abstract

The application discloses a detection method of polysaccharide substances in Tanreqing injection, and the detection method comprises the following steps: preparing and detecting the test sample solution and the dextran standard solution by a direct method, and preparing and detecting the test sample solution and the dextran standard solution by an indirect method; according to the detection results of the direct method and the indirect method, it is determined whether the polysaccharide substances exist in the Tanreqing injection, wherein the polysaccharide substances are dextran. The method is simple, efficient and rapid, and through the detection of the polysaccharide substances, a theoretical basis is provided for the quality control of the Tanreqing injection and the in-depth research of the pharmacodynamic substance basis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for detecting polysaccharides in Tanreqing injection. Background Technology

[0002] Tanreqing Injection is made from five medicinal herbs: Scutellaria baicalensis, bear bile powder, goat horn, honeysuckle, and forsythia. It has the effects of clearing heat, detoxifying, and resolving phlegm. It has significant efficacy in treating upper respiratory tract infections, acute bronchitis, pneumonia, pulmonary tuberculosis complicated with pulmonary infection, advanced lung cancer complicated with pulmonary infection, and severe asthma complicated with infection.

[0003] There are no reports in the existing technology regarding the detection methods of polysaccharides in Tanreqing injection. Summary of the Invention

[0004] Based on this, the present invention provides a method for detecting polysaccharides in Tanreqing injection, the method comprising the following steps:

[0005] The test solution and dextran standard solution were prepared and tested using a direct method.

[0006] The chromatographic conditions for detection in this direct method are as follows: a column packed with a mixed gel, a mobile phase of saline solution, a flow rate of 0.1–1.0 mL / min, a column temperature of 10–30 °C, a fluorescence detector with an excitation wavelength of 300–350 nm and an emission wavelength of 370–430 nm; and

[0007] The test solution and dextran standard solution were prepared and tested using an indirect method.

[0008] The chromatographic conditions for detection in this indirect method are as follows: a chromatographic column packed with octadecyl bonded silica gel is used; mobile phase A is selected from one or more of acetonitrile, methanol, and tetrahydrofuran; mobile phase B is an aqueous acid solution, an aqueous alkaline solution, and / or an aqueous buffer solution; the gradient elution program is: 0–2 min, 90% B, 2–30 min, 90→70% B; the flow rate is 0.5–1.5 ml / min; the column temperature is 10–30 °C; and the detection wavelength is 200–400 nm.

[0009] Based on the test results of the direct method and the indirect method, it was determined whether the Tanreqing injection contained polysaccharide substances, wherein the polysaccharide substances were glucan.

[0010] Furthermore, the Tanreqing injection is composed of Scutellaria baicalensis, bear bile powder, goat horn, honeysuckle, forsythia, and propylene glycol.

[0011] Furthermore, the steps for preparing the test solution using this direct method include:

[0012] (a) Take an appropriate amount of Tanreqing injection and purify it through a gel permeation chromatography column to obtain a polysaccharide sample solution;

[0013] (b) After drying the polysaccharide sample solution, add the coupling reagent solution, mix, and heat to obtain the first solution;

[0014] (c) Add a reducing agent solution to the first solution, mix, and heat to obtain a second solution; and

[0015] (d) Add water and phenol-chloroform solution to the second solution, centrifuge, discard the lower organic phase, add water and phenol-chloroform solution again, centrifuge, discard the lower organic phase, add water and phenol-chloroform solution a third time, centrifuge, discard the lower organic phase, and then filter the upper liquid through a membrane to obtain the test solution.

[0016] Furthermore, the steps for preparing the dextran standard solution by the direct method include: weighing an appropriate amount of dextran standard, dissolving it in water to obtain a dextran solution, and then proceeding with the same steps as steps (b) to (d) in the preparation method of the test sample solution to obtain the dextran standard solution.

[0017] Furthermore, steps (a) to (d) of the direct method for preparing the test solution include any one or more of the following items [1] to

[17] :

[0018] [1] The method of purifying Tanreqing injection in step (a) by gel permeation chromatography includes the following steps: take a gel permeation chromatography pre-packed column, shake it upside down to make the internal packing filler uniformly fill the inside of the pre-packed column, and minimize the generation of air bubbles; cut off the seal at the bottom of the pre-packed column, open the top cap of the pre-packed column, place it in a collection tube, centrifuge for the first time, discard the buffer solution obtained, then place the pre-packed column on another collection tube, add the Tanreqing injection to be purified on the column, centrifuge for the second time, and obtain the polysaccharide sample solution;

[0019] Furthermore, the gel is a polyacrylamide gel;

[0020] Furthermore, the gel permeation chromatography pre-packed column is a Bio-Spin 6Tris column;

[0021] Furthermore, the centrifuge speed is 2000–4000 r / min, for example, about 3000 r / min;

[0022] Furthermore, the time for this first centrifugation is 1 to 5 minutes, for example, about 2 minutes;

[0023] Furthermore, the second centrifugation time is 1 to 10 minutes, for example, about 4 minutes;

[0024] Furthermore, the volume of the Tanreqing injection is 50–150 μl, for example, about 100 μl;

[0025] Furthermore, the volume of the polysaccharide sample solution is 50–150 μl, for example, about 100 μl;

[0026] [2] The drying process in step (b) is nitrogen drying;

[0027] [3] The method for preparing the coupling reagent solution in step (b) is to dissolve 2-aminopyridine in glacial acetic acid to prepare a glacial acetic acid solution of 2500-3000 mg / ml, for example, about 2760 mg / ml of 2-aminopyridine.

[0028] [4] The volume ratio of the polysaccharide sample solution in step (a) to the coupling reagent solution in step (b) is (1 to 10):1, for example, about 5:1;

[0029] [5] The heating temperature in step (b) is 85°C to 95°C, for example, about 90°C;

[0030] [6] The heating time in step (b) is 50 min to 70 min, for example, about 60 min;

[0031] [7] The method for preparing the reducing agent solution in step (c) is to dissolve the boric acid 200-dimethylamine complex in acetic acid and water to prepare an acetic acid aqueous solution of boric acid 200-dimethylamine complex with a concentration of 1200-2000 mg / ml, for example, about 1538 mg / ml, wherein the volume ratio between acetic acid and water is about 8: about 5.

[0032] [8] The volume ratio of the polysaccharide sample solution in step (a) to the reducing agent solution in step (c) is (5-12):7, for example, about 10:7;

[0033] [9] The heating temperature in step (c) is 75°C to 85°C, for example, about 80°C;

[0034]

[10] The heating time in step (c) is 25 to 45 minutes, for example, about 35 minutes;

[0035]

[11] The volume ratio of the polysaccharide sample solution in step (a) to the water in step (d) is (5-12):9, for example, about 10:9;

[0036]

[12] The volume ratio of the phenol-chloroform solution in step (d) is (0.1-2):(0.1-2), for example, about 1:about 1;

[0037]

[13] The volume ratio of the polysaccharide sample solution in step (a) to the phenol-chloroform solution in step (d) is (5-12):9, for example, about 10:9;

[0038]

[14] The volume ratio of the water to the phenol-chloroform solution in step (d) is (0.1-2):(0.1-2), for example, about 1:about 1;

[0039]

[15] The centrifuge speed in step (d) is 5000 to 7000 r / min, for example about 6000 r / min;

[0040]

[16] The centrifugation time in step (d) is 30 to 90 seconds, for example, about 60 seconds;

[0041]

[17] The pore size of the filter membrane in step (d) is 0.45 μm.

[0042] Furthermore, the steps for preparing the test solution using this indirect method include:

[0043] (A) Take an appropriate amount of Tanreqing injection and purify it through a gel permeation chromatography column to obtain a polysaccharide sample solution;

[0044] (B) Add trifluoroacetic acid solution to the polysaccharide sample solution, vortex, hydrolyze, cool, and dry to obtain the first sample;

[0045] (C) Add water to the first sample to make up the total volume, then add ammonia solution and PMP methanol solution, vortex, react, cool, and dry to obtain the second sample; and

[0046] (D) Add KH2PO4 solution and chloroform to the second sample, vortex, let stand, discard the lower organic phase, add chloroform again, vortex, let stand, discard the lower organic phase, add chloroform a third time, vortex, let stand, discard the lower organic phase, and then filter the upper liquid through a membrane to obtain the test solution.

[0047] Furthermore, the steps for preparing the dextran standard solution by the indirect method include: weighing an appropriate amount of dextran standard, dissolving it in water to obtain a dextran solution, and then proceeding with the same steps (C) to (D) as in the preparation method of the test sample solution to obtain the dextran standard solution.

[0048] Furthermore, steps (A) to (D) of the indirect method for preparing the test solution include any one or more of the following items [1] to

[21] :

[0049] [1] The method of purifying Tanreqing injection in step (A) by gel permeation chromatography includes the following steps: take a gel permeation chromatography pre-packed column, shake it upside down to make the internal packing filler uniformly fill the inside of the pre-packed column, and minimize the generation of air bubbles; cut off the seal at the bottom of the pre-packed column, open the top cap of the pre-packed column, place it in a collection tube, centrifuge for the first time, discard the buffer solution obtained, then place the pre-packed column on another collection tube, add the Tanreqing injection to be purified on the column, centrifuge for the second time, and obtain the polysaccharide sample solution;

[0050] Furthermore, the gel is a polyacrylamide gel;

[0051] Furthermore, the gel permeation chromatography pre-packed column is a Bio-Spin 6Tris column;

[0052] Furthermore, the centrifuge speed is 2000–4000 r / min, for example, about 3000 r / min;

[0053] Furthermore, the time for this first centrifugation is 1 to 5 minutes, for example, about 2 minutes;

[0054] Furthermore, the second centrifugation time is 1 to 10 minutes, for example, about 4 minutes;

[0055] Furthermore, the volume of the Tanreqing injection is 50–150 μl, for example, about 100 μl;

[0056] Furthermore, the volume of the polysaccharide sample solution is 50–150 μl, for example, about 100 μl;

[0057] [2] The molar concentration of the trifluoroacetic acid in step (B) is 2 to 6 mol / L, for example, about 4 mol / L;

[0058] [3] The volume ratio of the polysaccharide sample solution in step (A) to the trifluoroacetic acid in step (B) is (0.5-5):1, for example, about 1:1;

[0059] [4] The duration of the vortex in step (B) is 10 to 60 seconds, for example, about 30 seconds;

[0060] [5] The temperature of the hydrolysis in step (B) is 90 to 130°C, for example, about 110°C;

[0061] [6] The hydrolysis time in step (B) is 30 to 90 minutes, for example, about 60 minutes;

[0062] [7] The drying in step (B) or step (C) is nitrogen drying;

[0063] [8] The volume ratio of the polysaccharide sample solution in step (A) to the total amount of water added in step (C) is (0.5 to 5):1, for example, about 1:1;

[0064] [9] The volume percentage concentration of the ammonia solution in step (C) is 10% to 30%, for example, about 20%;

[0065]

[10] The volume ratio of the polysaccharide sample solution in step (A) to the ammonia solution in step (C) is (0.5 to 5):1, for example, about 1:1;

[0066]

[11] The molar concentration of the PMP methanol solution in step (C) is 0.2 to 0.8 mol / L, for example, about 0.5 mol / L;

[0067]

[12] The volume ratio of the polysaccharide sample solution in step (A) to the PMP methanol solution in step (C) is (0.5 to 5):1, for example, about 1:1;

[0068]

[13] The duration of the vortex in step (C) is 10 to 60 seconds, for example, about 30 seconds;

[0069]

[14] The temperature of the reaction in step (C) is 50 to 90°C, for example, about 70°C;

[0070]

[15] The reaction time in step (C) is 30 to 90 minutes, for example, about 60 minutes;

[0071]

[16] The molar concentration of the KH2PO4 solution in step (D) is 0.01 to 0.2 mol / L, for example, about 0.1 mol / L;

[0072]

[17] The volume ratio of the polysaccharide sample solution in step (A) to the KH2PO4 solution in step (D) is 1:(15-25), for example, 1:about 20;

[0073]

[18] The volume ratio of the polysaccharide sample solution in step (A) to the chloroform in step (D) is 1:(15-25), for example, 1:about 20;

[0074]

[19] The duration of the vortex in step (D) is 10 to 60 seconds, for example, about 30 seconds;

[0075]

[20] The settling time in step (D) is 2 to 8 minutes, for example, about 5 minutes;

[0076]

[21] The pore size of the filter membrane in step (D) is 0.45 μm.

[0077] Furthermore, the aqueous acid solution, aqueous alkaline solution, and / or aqueous buffer salt solution are selected from one or more weak acids and their salts, weak bases and their salts of different concentrations.

[0078] Furthermore, the aqueous acid solution, aqueous alkaline solution, and / or aqueous buffer solution are selected from different concentrations of formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid.

[0079] Furthermore, the acid aqueous solution is an acid aqueous solution of 0.005% to 0.12%.

[0080] Furthermore, the buffer salt solution is an aqueous solution of phosphate and / or an aqueous solution of acetate.

[0081] Furthermore, the pH value of the buffer salt solution is no greater than 7.2, for example, about 6.8.

[0082] Furthermore, the detection wavelength is 220–280 nm, for example, 250 nm.

[0083] Furthermore, the peak purity of the chromatographic peak corresponding to this reference standard is greater than 995.

[0084] Furthermore, the resolution of the chromatographic peak corresponding to this reference standard is greater than 2.0.

[0085] Furthermore, the exclusion limit of this hybrid gel is 7 × 10⁻⁶. 6 .

[0086] Furthermore, the polysaccharides detected in the Tanreqing injection were plant polysaccharides with a molecular weight greater than or equal to 5000 Da.

[0087] Furthermore, the salt solution is an aqueous solution of sodium nitrate.

[0088] Furthermore, the molar concentration of the sodium nitrate aqueous solution is 0.01 to 1.0 mol / L, for example, about 0.1 mol / L.

[0089] Furthermore, the flow rate is 0.2–0.6 ml / min, for example, about 0.4 ml / min.

[0090] Furthermore, the excitation wavelength is 320–350 nm, for example, about 330 nm.

[0091] Furthermore, the emission wavelength is 375–425 nm, for example, about 400 nm.

[0092] Furthermore, the standard for identifying whether the Tanreqing injection contains polysaccharides includes: comparing the chromatogram of the test solution with the chromatogram of the polysaccharide standard solution to check whether there is a corresponding chromatographic peak at the position of the polysaccharide chromatographic peak in the test solution; if there is no corresponding chromatographic peak in both the direct method and the indirect method, then the Tanreqing injection does not contain polysaccharides, wherein the polysaccharide is dextran.

[0093] According to another aspect of the present invention, the above-described detection method is provided for use in the quality detection and / or quality evaluation and / or quality control of polysaccharides in Tanreqing injection.

[0094] The beneficial effects of this invention are:

[0095] The method of this invention is simple, efficient and rapid. By detecting polysaccharide substances, it provides a theoretical basis for the quality control of Tanreqing injection and in-depth research on the pharmacodynamic material basis. Attached Figure Description

[0096] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0097] Figure 1 The HPLC chromatograms (differential refractive index detector) of a 12000 Da dextran standard before and after purification are shown.

[0098] Figure 2 The HPLC chromatograms (differential refractive index detector) of a 5000 Da dextran standard before and after purification are shown.

[0099] Figure 3 The chromatograms (differential refractive index detector) of a 1000 Da dextran standard before and after purification are shown.

[0100] Figure 4 The images show the high-performance liquid chromatograms (HPLC) of baicalin in Tanreqing injection before and after polyacrylamide gel purification. The top image shows the baicalin peak (before purification, diluted 100 times), and the bottom image shows the baicalin peak (after purification, undiluted).

[0101] Figure 5 The images show high-performance liquid chromatograms (HPLC) of organic acid components in Tanreqing injection before and after purification with polyacrylamide gel electrophoresis. The top image shows the organic acid components (before purification), and the bottom image shows the organic acid components (after purification, chromatogram magnified 10 times). Detailed Implementation

[0102] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0103] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0104] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0105] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0106] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0107] As described in the background section, there are no reports in the prior art regarding methods for detecting polysaccharides in Tanreqing injection. To address the above problem, this invention provides a method for detecting polysaccharides in Tanreqing injection, which includes the following steps:

[0108] The test solution and dextran standard solution were prepared and tested using a direct method.

[0109] The chromatographic conditions for detection in this direct method are as follows: a column packed with a mixed gel, a mobile phase of saline solution, a flow rate of 0.1–1.0 mL / min, a column temperature of 10–30 °C, a fluorescence detector with an excitation wavelength of 300–350 nm and an emission wavelength of 370–430 nm; and

[0110] The test solution and dextran standard solution were prepared and tested using an indirect method.

[0111] The chromatographic conditions for detection in this indirect method are as follows: a chromatographic column packed with octadecyl bonded silica gel is used; mobile phase A is selected from one or more of acetonitrile, methanol, and tetrahydrofuran; mobile phase B is an aqueous acid solution, an aqueous alkaline solution, and / or an aqueous buffer solution; the gradient elution program is: 0–2 min, 90% B, 2–30 min, 90→70% B; the flow rate is 0.5–1.5 ml / min; the column temperature is 10–30 °C; and the detection wavelength is 200–400 nm.

[0112] Based on the test results of the direct method and the indirect method, it was determined whether the Tanreqing injection contained polysaccharide substances, wherein the polysaccharide substances were glucan.

[0113] In this invention, when temperature, rate, time, concentration, injection volume, equivalent, concentration, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “10–30” is disclosed, the described range should be interpreted as including ranges “10–30”, “10–28”, “10–25”, “10–23”, “10–20”, “10–15”, “10–13”, “15–30”, “15–28”, “15–25”, “15–20”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0114] To avoid adverse reactions to the human body caused by the macromolecular substances contained in traditional Chinese medicine injections during use, this invention has explored in depth the detection method of polysaccharides in Tanreqing injection, aiming to effectively control the presence of macromolecular polysaccharides in the injection. The method established in this invention has been applied to multiple batches of Tanreqing injection samples with different storage periods, and no relevant macromolecular polysaccharides were detected in any of the results.

[0115] In a preferred embodiment, the Tanreqing injection is composed of Scutellaria baicalensis, bear bile powder, goat horn, honeysuckle, forsythia, and propylene glycol.

[0116] In a preferred embodiment, the steps of the direct method for preparing the test solution include:

[0117] (a) Take an appropriate amount of Tanreqing injection and purify it through a gel permeation chromatography column to obtain a polysaccharide sample solution;

[0118] (b) After drying the polysaccharide sample solution, add the coupling reagent solution, mix, and heat to obtain the first solution;

[0119] (c) Add a reducing agent solution to the first solution, mix, and heat to obtain a second solution; and

[0120] (d) Add water and phenol-chloroform solution to the second solution, centrifuge, discard the lower organic phase, add water and phenol-chloroform solution again, centrifuge, discard the lower organic phase, add water and phenol-chloroform solution a third time, centrifuge, discard the lower organic phase, and then filter the upper liquid through a membrane to obtain the test solution.

[0121] In a preferred embodiment, the steps of preparing the dextran standard solution by the direct method include: weighing an appropriate amount of dextran standard, dissolving it in water to obtain a dextran solution, and then proceeding with the same steps (b) to (d) as in the preparation method of the test sample solution to obtain the dextran standard solution.

[0122] In a preferred embodiment, steps (a) to (d) of the direct method for preparing the test solution include any one or more of the following items [1] to

[17] :

[0123] [1] The method of purifying Tanreqing injection in step (a) by gel permeation chromatography includes the following steps: take a gel permeation chromatography pre-packed column, shake it upside down to make the internal packing filler uniformly fill the inside of the pre-packed column, and minimize the generation of air bubbles; cut off the seal at the bottom of the pre-packed column, open the top cap of the pre-packed column, place it in a collection tube, centrifuge for the first time, discard the buffer solution obtained, then place the pre-packed column on another collection tube, add the Tanreqing injection to be purified on the column, centrifuge for the second time, and obtain the polysaccharide sample solution;

[0124] In a preferred embodiment, the gel is a polyacrylamide gel;

[0125] In a preferred embodiment, the gel permeation chromatography pre-packed column is a Bio-Spin 6Tris column;

[0126] In a preferred embodiment, the centrifugation speed is 2000 to 4000 r / min, for example, about 3000 r / min;

[0127] In a preferred embodiment, the first centrifugation time is 1 to 5 minutes, for example, about 2 minutes;

[0128] In a preferred embodiment, the second centrifugation time is 1 to 10 minutes, for example, about 4 minutes;

[0129] In a preferred embodiment, the volume of the Tanreqing injection is 50–150 μl, for example, about 100 μl;

[0130] In a preferred embodiment, the volume of the polysaccharide sample solution is 50–150 μl, for example, about 100 μl;

[0131] [2] The drying process in step (b) is nitrogen drying;

[0132] [3] The method for preparing the coupling reagent solution in step (b) is to dissolve 2-aminopyridine in glacial acetic acid to prepare a glacial acetic acid solution of 2500-3000 mg / ml, for example, about 2760 mg / ml of 2-aminopyridine.

[0133] [4] The volume ratio of the polysaccharide sample solution in step (a) to the coupling reagent solution in step (b) is (1 to 10):1, for example, about 5:1;

[0134] [5] The heating temperature in step (b) is 85°C to 95°C, for example, about 90°C;

[0135] [6] The heating time in step (b) is 50 min to 70 min, for example, about 60 min;

[0136] [7] The method for preparing the reducing agent solution in step (c) is to dissolve the boric acid 200-dimethylamine complex in acetic acid and water to prepare an acetic acid aqueous solution of boric acid 200-dimethylamine complex with a concentration of 1200-2000 mg / ml, for example, about 1538 mg / ml, wherein the volume ratio between acetic acid and water is about 8: about 5.

[0137] [8] The volume ratio of the polysaccharide sample solution in step (a) to the reducing agent solution in step (c) is (5-12):7, for example, about 10:7;

[0138] [9] The heating temperature in step (c) is 75°C to 85°C, for example, about 80°C;

[0139]

[10] The heating time in step (c) is 25 to 45 minutes, for example, about 35 minutes;

[0140]

[11] The volume ratio of the polysaccharide sample solution in step (a) to the water in step (d) is (5-12):9, for example, about 10:9;

[0141]

[12] The volume ratio of the phenol-chloroform solution in step (d) is (0.1-2):(0.1-2), for example, about 1:about 1;

[0142]

[13] The volume ratio of the polysaccharide sample solution in step (a) to the phenol-chloroform solution in step (d) is (5-12):9, for example, about 10:9;

[0143]

[14] The volume ratio of the water to the phenol-chloroform solution in step (d) is (0.1-2):(0.1-2), for example, about 1:about 1;

[0144]

[15] The centrifuge speed in step (d) is 5000 to 7000 r / min, for example about 6000 r / min;

[0145]

[16] The centrifugation time in step (d) is 30 to 90 seconds, for example, about 60 seconds;

[0146]

[17] The pore size of the filter membrane in step (d) is 0.45 μm.

[0147] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3000" includes ±5% of 3000, or from 2850 to 3150; "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 4" includes ±5% of 4, or from 3.8 to 4.2; "about 100" includes ±5% of 100, or from 95 to 105; "about 2760" includes ±5% of 2760, or from 2622 to 2898; "about 5" includes ±5% of 5, or from 4.75 to 5.25; "about 90" includes ±5% of 90, or from 85.5 to 94.5; "about 60" includes ±5% of 60. %, or from 57 to 63; “about 1538” includes 1538 ± 5%, or from 1461.1 to 1614.9; “about 8” includes 8 ± 5%, or from 7.6 to 8.4; “about 10” includes 10 ± 5%, or from 9.5 to 10.5; “about 80” includes 80 ± 5%, or from 76 to 84; “about 35” includes 35 ± 5%, or from 33.25 to 36.75; “about 1” includes 1 ± 5%, or from 0.95 to 1.05; “about 6000” includes 6000 ± 5%, or from 5700 to 6300.

[0148] In a preferred embodiment, the indirect method for preparing the test solution includes the following steps:

[0149] (A) Take an appropriate amount of Tanreqing injection and purify it through a gel permeation chromatography column to obtain a polysaccharide sample solution;

[0150] (B) Add trifluoroacetic acid solution to the polysaccharide sample solution, vortex, hydrolyze, cool, and dry to obtain the first sample;

[0151] (C) Add water to the first sample to make up the total volume, then add ammonia solution and PMP methanol solution, vortex, react, cool, and dry to obtain the second sample; and

[0152] (D) Add KH2PO4 solution and chloroform to the second sample, vortex, let stand, discard the lower organic phase, add chloroform again, vortex, let stand, discard the lower organic phase, add chloroform a third time, vortex, let stand, discard the lower organic phase, and then filter the upper liquid through a membrane to obtain the test solution.

[0153] In a preferred embodiment, the indirect method for preparing the dextran standard solution includes: weighing an appropriate amount of dextran standard, dissolving it in water to obtain a dextran solution, and then proceeding with the same steps (C) to (D) as in the preparation method of the test sample solution to obtain the dextran standard solution.

[0154] In a preferred embodiment, steps (A) to (D) of the indirect method for preparing the test solution include any one or more of the following items [1] to

[21] :

[0155] [1] The method of purifying Tanreqing injection in step (A) by gel permeation chromatography includes the following steps: take a gel permeation chromatography pre-packed column, shake it upside down to make the internal packing filler uniformly fill the inside of the pre-packed column, and minimize the generation of air bubbles; cut off the seal at the bottom of the pre-packed column, open the top cap of the pre-packed column, place it in a collection tube, centrifuge for the first time, discard the buffer solution obtained, then place the pre-packed column on another collection tube, add the Tanreqing injection to be purified on the column, centrifuge for the second time, and obtain the polysaccharide sample solution;

[0156] In a preferred embodiment, the gel is a polyacrylamide gel;

[0157] In a preferred embodiment, the gel permeation chromatography pre-packed column is a Bio-Spin 6Tris column;

[0158] In a preferred embodiment, the centrifugation speed is 2000 to 4000 r / min, for example, about 3000 r / min;

[0159] In a preferred embodiment, the first centrifugation time is 1 to 5 minutes, for example, about 2 minutes;

[0160] In a preferred embodiment, the second centrifugation time is 1 to 10 minutes, for example, about 4 minutes;

[0161] In a preferred embodiment, the volume of the Tanreqing injection is 50–150 μl, for example, about 100 μl;

[0162] In a preferred embodiment, the volume of the polysaccharide sample solution is 50–150 μl, for example, about 100 μl;

[0163] [2] The molar concentration of the trifluoroacetic acid in step (B) is 2 to 6 mol / L, for example, about 4 mol / L;

[0164] [3] The volume ratio of the polysaccharide sample solution in step (A) to the trifluoroacetic acid in step (B) is (0.5-5):1, for example, about 1:1;

[0165] [4] The duration of the vortex in step (B) is 10 to 60 seconds, for example, about 30 seconds;

[0166] [5] The temperature of the hydrolysis in step (B) is 90 to 130°C, for example, about 110°C;

[0167] [6] The hydrolysis time in step (B) is 30 to 90 minutes, for example, about 60 minutes;

[0168] [7] The drying in step (B) or step (C) is nitrogen drying;

[0169] [8] The volume ratio of the polysaccharide sample solution in step (A) to the total amount of water added in step (C) is (0.5 to 5):1, for example, about 1:1;

[0170] [9] The volume percentage concentration of the ammonia solution in step (C) is 10% to 30%, for example, about 20%;

[0171]

[10] The volume ratio of the polysaccharide sample solution in step (A) to the ammonia solution in step (C) is (0.5 to 5):1, for example, about 1:1;

[0172]

[11] The molar concentration of the PMP methanol solution in step (C) is 0.2 to 0.8 mol / L, for example, about 0.5 mol / L;

[0173]

[12] The volume ratio of the polysaccharide sample solution in step (A) to the PMP methanol solution in step (C) is (0.5 to 5):1, for example, about 1:1;

[0174]

[13] The duration of the vortex in step (C) is 10 to 60 seconds, for example, about 30 seconds;

[0175]

[14] The temperature of the reaction in step (C) is 50 to 90°C, for example, about 70°C;

[0176]

[15] The reaction time in step (C) is 30 to 90 minutes, for example, about 60 minutes;

[0177]

[16] The molar concentration of the KH2PO4 solution in step (D) is 0.01 to 0.2 mol / L, for example, about 0.1 mol / L;

[0178]

[17] The volume ratio of the polysaccharide sample solution in step (A) to the KH2PO4 solution in step (D) is 1:(15-25), for example, 1:about 20;

[0179]

[18] The volume ratio of the polysaccharide sample solution in step (A) to the chloroform in step (D) is 1:(15-25), for example, 1:about 20;

[0180]

[19] The duration of the vortex in step (D) is 10 to 60 seconds, for example, about 30 seconds;

[0181]

[20] The settling time in step (D) is 2 to 8 minutes, for example, about 5 minutes;

[0182]

[21] The pore size of the filter membrane in step (D) is 0.45 μm.

[0183] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3000" includes ±5% of 3000, or from 2850 to 3150; "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 4" includes ±5% of 4, or from 3.8 to 4.2; "about 100" includes ±5% of 100, or from 95 to 105; "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 30" includes ±5% of 30, or from 28.5 to 31.5; "about 110" includes ±5% of 110, or from 104.5 to 115.5; "about 60" includes ±5% of 60, or from 57 to 63; "About 20" includes 20 ± 5%, or from 19 to 21; "About 0.5" includes 0.5 ± 5%, or from 0.475 to 0.525; "About 30" includes 30 ± 5%, or from 28.5 to 31.5; "About 70" includes 70 ± 5%, or from 67.5 to 73.5; "About 0.1" includes 0.1 ± 5%, or from 0.095 to 0.105; "About 20" includes 20 ± 5%, or from 19 to 21; "About 30" includes 30 ± 5%, or from 28.5 to 31.5; "About 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0184] In a preferred embodiment, the acid aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution are selected from one or more weak acids and their salts, and weak bases and their salts of different concentrations.

[0185] In a preferred embodiment, the acidic aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution is selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid.

[0186] In a preferred embodiment, the acidic aqueous solution is a 0.005% to 0.12% acidic aqueous solution.

[0187] In a preferred embodiment, the buffer salt solution is an aqueous solution of phosphate and / or an aqueous solution of acetate.

[0188] In a preferred embodiment, the pH value of the buffer salt solution is not greater than 7.2, for example, about 6.8.

[0189] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6.8" includes ±5% of 6.8, or from 6.46 to 7.14.

[0190] In a preferred embodiment, the detection wavelength is 220–280 nm, for example, 250 nm.

[0191] In a preferred embodiment, the peak purity of the chromatographic peak corresponding to the reference standard is greater than 995.

[0192] In a preferred embodiment, the resolution of the chromatographic peak corresponding to the reference standard is greater than 2.0.

[0193] In a preferred embodiment, the exclusion limit of the hybrid gel is 7 × 10⁻⁶. 6 .

[0194] In a preferred embodiment, the polysaccharide detected in the Tanreqing injection is a plant polysaccharide with a molecular weight greater than or equal to 5000 Da.

[0195] In a preferred embodiment, the salt solution is an aqueous solution of sodium nitrate.

[0196] In a preferred embodiment, the molar concentration of the sodium nitrate aqueous solution is 0.01 to 1.0 mol / L, for example, about 0.1 mol / L.

[0197] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

[0198] In a preferred embodiment, the flow rate is 0.2 to 0.6 ml / min, for example, about 0.4 ml / min.

[0199] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.4" includes ±5% of 0.4, or from 0.38 to 0.42.

[0200] In a preferred embodiment, the excitation wavelength is 320–350 nm, for example, about 330 nm.

[0201] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 330" includes ±5% of 330, or from 313.5 to 346.5.

[0202] In a preferred embodiment, the emission wavelength is 375–425 nm, for example, about 400 nm.

[0203] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 400" includes ±5% of 400, or from 380 to 420.

[0204] In a preferred embodiment, the standard for identifying whether the Tanreqing injection contains polysaccharides includes: comparing the chromatogram of the test solution with the chromatogram of the polysaccharide standard solution to see if there is a corresponding chromatographic peak at the position of the polysaccharide chromatographic peak in the test solution; if there is no corresponding chromatographic peak in both the direct method and the indirect method, then the Tanreqing injection does not contain polysaccharides, wherein the polysaccharide is dextran.

[0205] According to another aspect of the present invention, the above-described detection method is provided for use in the quality detection and / or quality evaluation and / or quality control of polysaccharides in Tanreqing injection.

[0206] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0207] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0208] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0209] Example

[0210] 1. Instruments and reagents

[0211] An Agilent 1100 liquid chromatograph equipped with a diode array detector and a differential refractive index detector (Agilent Technologies, USA); and an Agilent 6410 triple quadrupole tandem mass spectrometer (Agilent Technologies, USA). Bio-Spin 6Tris columns (pre-packed polyacrylamide gel column, molecular weight cutoff 5000 Da) were purchased from Bio-Rad Laboratories, USA; derivatization reagents PMP, 2-aminopyridine, and 200-dimethylamine borate complex were provided by Sigma-Aldrich, USA; monosaccharide standards and dextran standards were also purchased from Sigma-Aldrich, USA; the Tanreqing injection sample was provided by Shanghai Kaibao Pharmaceutical Co., Ltd. (as shown in Table 1); all other reagents were of analytical grade and sourced from Sinopharm Chemical Reagent Co., Ltd.

[0212] Table 1 Sample List of Tanreqing Injection

[0213]

[0214]

[0215] 2. Source analysis of polysaccharide components in Tanreqing Injection

[0216] Polysaccharides are macromolecules composed of numerous small monosaccharide molecules linked together by covalent bonds. Polymers composed of only a few monosaccharides (from 5 to 10) are generally called oligosaccharides. In this regard, a polysaccharide macromolecule typically has a molar mass greater than approximately 1000–5000 Da, but there is no strict lower limit definition. The distinction between oligosaccharides and polysaccharides remains ambiguous because, in most aspects of interest, there is no abrupt change between the two. This invention primarily focuses on polysaccharides with a molecular weight greater than 5000 Da.

[0217] The prescription for Tanreqing Injection consists of three herbal medicines and two animal medicines: Scutellaria baicalensis, Lonicera japonica, Forsythia suspensa, bear bile powder, and goat horn. Literature reports that Scutellaria baicalensis and Lonicera japonica contain polysaccharide components. Forsythia suspensa, as a herbal medicine, must also contain some plant polysaccharides; however, no reports have been found regarding polysaccharide components in the two animal medicines.

[0218] In terms of preparation process, all three herbal medicines are extracted by water decoction, which is more suitable for the dissolution of polysaccharide components. In the extraction process of honeysuckle, the addition of strong acids and alkalis may cause certain hydrolysis and destruction of sugar chains.

[0219] In summary, based on relevant literature, the polysaccharide components that may be present in Tanreqing Injection are mainly plant polysaccharides, generally composed of arabinose, xylose, glucose, glucuronic acid, etc., with fewer glycosylated proteins after protein expression, such as N-linked oligosaccharides and O-linked oligosaccharides. Therefore, the research object of this invention is the plant polysaccharides with a molecular weight greater than 5000 Da in Tanreqing Injection, and simultaneously, starting from the source, the plant polysaccharides that may be contained in the three intermediate products (honeysuckle extract, forsythia extract, and scutellaria extract) in the preparation process of Tanreqing Injection are investigated.

[0220] 3. Research Methods and Results

[0221] 3.1 Research on extraction and purification methods for polysaccharide components

[0222] 3.1.1 Extraction Method

[0223] Based on the preparation process of Tanreqing Injection, the polysaccharide components that may be contained in its intermediates or injection should be water-soluble plant polysaccharides; since Tanreqing Injection is administered in the form of an aqueous solution, the extraction method can be disregarded, and direct sampling is sufficient.

[0224] 3.1.2 Purification Method

[0225] Purification of polysaccharides is necessary for both chromatographic and mass spectrometric analyses. For example, when analyzing sugar composition, monosaccharide components in the sample solution can interfere with the determination. For mass spectrometry, the ionization of carbohydrate compounds is more susceptible to interference from salts than that of proteins. Therefore, proper purification is essential.

[0226] Currently, the main purification techniques for polysaccharides include dialysis and solid-phase extraction. Dialysis is simple to operate, and small molecules such as salts in polysaccharides can be directly removed with aqueous solutions. However, dialysis is time-consuming and not suitable for routine quality analysis.

[0227] Solid phase extraction technology has a wide range of applications and a variety of fillers are available, such as graphitized carbon, polyacrylamide gel, and perfluorosulfonic acid resin.

[0228] Polyacrylamide gel is a synthetic gel made by cross-linking acrylamide with acrylamide as the unit. It is dried, pulverized, or shaped into granules. Various types of gels can be produced by controlling the amount of cross-linking agent. It primarily separates compounds using the principle of molecular sieves. Because this packing material is well-suited to aqueous environments, it is particularly suitable for separating water-soluble proteins and polysaccharides.

[0229] Taking all factors into consideration, this invention uses polyacrylamide gel (molecular weight cutoff 5000 Da) and examines the purification method and its effectiveness.

[0230] 3.1.2.1 Tests on dextran standards of different molecular weights

[0231] Dextran standards with molecular weights of 1000, 5000, and 12000 were used as reference materials, and the high-performance liquid chromatography (HPLC) chromatograms of the standard solutions before and after passing through a polyacrylamide gel (molecular weight cutoff of 5000 Da) were compared. The results showed that the dextran standard with a molecular weight of 12000 Da could completely pass through the polyacrylamide gel, the dextran standard with a molecular weight of 5000 Da, due to its proximity to the size exclusion limit of the gel column, also largely passed through the purification process, while the dextran standard with a molecular weight of 1000 Da was completely retained in the gel column. Figure 1 , Figure 2 and Figure 3 As shown.

[0232] 3.1.2.2 Purification effect of small molecule substances in Tanreqing Injection

[0233] The study also investigated the removal efficiency of polyacrylamide gel on small molecules in Tanreqing injection. Results showed that the amount of baicalin remaining in the test solution after purification with polyacrylamide gel was 0.02% of the original amount. Furthermore, the purification effect was further verified using a method for detecting organic acids in Tanreqing injection. Results are as follows... Figure 4 and Figure 5 As shown.

[0234] The above results indicate that after purification with polyacrylamide gel, only a small amount of baicalin was detected in Tanreqing injection, while the remaining components were effectively removed. This demonstrates that most small molecule compounds in the test solution can be effectively removed after purification with polyacrylamide gel.

[0235] 3.1.2.3 Purification test of the sampled solution

[0236] Approximately 10 mg of dextran standards with molecular weights of 5000 Da and 12000 Da were weighed and dissolved in 10 ml of Tanreqing injection solution. The content of polysaccharide components in the samples before and after purification was compared. The results showed that there was no significant loss of polysaccharide components in the sample solutions after purification with polyacrylamide gel.

[0237] 3.2 Research on methods for detecting polysaccharide components

[0238] 3.2.1 Overview

[0239] The separation of carbohydrate compounds generally employs chromatographic techniques, such as gas chromatography (GC), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC), and capillary electrophoresis (CE). In recent years, HPLC and CE have seen the most rapid development, offering advantages such as high separation efficiency, short analysis time, small injection volume, low detection limits, and multi-mode processing. However, the detection of carbohydrates is challenging because they lack strong UV absorption and fluorescent groups, and are typically electronegative. Therefore, the detection of carbohydrates usually requires derivatization to impart UV absorption, fluorescence, or other measurable physicochemical properties.

[0240] This invention employs the classic reductive amine method for the derivatization and labeling of polysaccharide compounds. In this two-step derivatization method, the terminal carbonyl group in the sample first reacts with a primary amine to generate an imine, which is then reduced to the product. The derivatizing reagent is the most commonly used 2-aminopyridine. The determination is then performed using high-performance liquid chromatography with a fluorescence detector, and necessary structural analysis is conducted using mass spectrometry. Simultaneously, a method for the acid hydrolysis derivatization of polysaccharides to determine monosaccharides was also established to obtain greater detection sensitivity. Trifluoroacetic acid is used for hydrolysis, and the hydrolysis product is then derivatized and labeled using PMP. The determination is then performed using high-performance liquid chromatography with a UV detector.

[0241] The specific method is as follows:

[0242] Gel permeation pre-packed column purification: Take a Bio-Spin 6Tris column, invert and shake it to ensure the internal packing material is evenly filled inside the column, minimizing the generation of air bubbles; cut off the seal at the bottom of the pre-packed column, open the top cap of the pre-packed column, place it in a collection tube, centrifuge at 3000 rpm for 2 minutes, discard the resulting buffer solution, then place the pre-packed column on another collection tube, add 100 μL of the sample solution to be purified to the column, centrifuge at 3000 rpm for 4 minutes, and the solution is ready.

[0243] Polysaccharide derivatization (direct method): 100 μL of polysaccharide sample solution purified by gel permeation chromatography was injected into a 2.0 mL conical microtube, and the sample was dried under nitrogen. 0.02 mL of coupling reagent solution (552 mg 2-aminopyridine dissolved in 0.2 mL glacial acetic acid) was added, and the coupling reagent solution and sample were mixed. The mixture was heated at 90 °C for 1 h. 0.07 mL of reducing agent solution (200 mg boric acid-200-dimethylamine complex dissolved in 0.08 mL acetic acid and 0.05 mL water) was added. The reducing reagent solution and sample were mixed, and the mixture was heated at 80 °C for 35 min. 0.09 mL of water and 0.09 mL of phenol / chloroform solution (1:1) were added to the reaction solution, and the mixture was centrifuged at 6000 rpm for 1 min. The lower organic phase was discarded. Repeat the operation twice starting from "Add 0.09 ml of water..." On the last run, filter the supernatant through a 0.45 μm filter membrane and transfer it to a new vial for analysis by reversed-phase high-performance liquid chromatography and mass spectrometry.

[0244] Liquid chromatography conditions: Gel permeation chromatography packing material was used as the stationary phase (mixed gel packing material with an exclusion limit of 7×10⁶, mainly used for analyzing polysaccharides with a molecular weight of 5000 Da or higher); 100% 0.1M sodium nitrate was used as the mobile phase, and the flow rate was 0.4 ml / min.

[0245] Fluorescence detector, excitation wavelength 330nm, emission wavelength 400nm.

[0246] Acid hydrolysis of polysaccharides + monosaccharide derivatization (indirect method): Take 100 μL of polysaccharide sample solution purified by gel permeation chromatography into a 2.0 mL vial, add 100 μL of 4 mol / L trifluoroacetic acid solution, seal, vortex for 30 s, and place in a 110 °C oven for 1 h of hydrolysis. After cooling, open the cap and dry with nitrogen to obtain the hydrolyzed sample. Take the above hydrolyzed sample, add water to make up to 100 μL, add 100 μL of 20% ammonia solution and 100 μL of 0.5 mol / L PMP methanol solution into a 2 mL brown vial, cap, vortex for 30 s, react at 70 °C for 1 h, cool to room temperature, dry with nitrogen, add 2 mL of 0.1 mol / L KH2PO4 solution, add 2 mL of chloroform (chromatographic grade), vortex for 30 s, let stand for 5 min, and discard the lower layer. Repeat the procedure twice, starting with "add 2 ml chloroform". On the last run, filter the supernatant through a 0.45 μm filter membrane and transfer it to a new vial for reversed-phase high-performance liquid chromatography (RP-HPLC) analysis.

[0247] Liquid chromatography conditions: Octadecyl-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A, and phosphate buffer (pH 6.8) was used as mobile phase B, with gradient elution according to Table 2. The detection wavelength was 250 nm.

[0248] Table 2 Gradient elution process

[0249] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～2 10 90 2～30 10→30 90→70

[0250] Of the methods described above, the direct method is more specific and accurate, and can be used for the structural analysis and sequencing of polysaccharide components, but its sensitivity is slightly lower. The indirect method, due to the hydrolysis step, can interfere with the detection results if the sample solution contains glycosides (such as baicalin), but its detection sensitivity is even lower and it can be used to determine the monosaccharide composition of polysaccharides. Therefore, this study combines the two methods, taking advantage of their respective strengths.

[0251] 3.2.2 Qualitative and quantitative analysis of polysaccharide components in Tanreqing injection and its intermediate extract

[0252] 3.2.2.1 Direct method (pyridine amination of polysaccharides)

[0253] ① Analysis of polysaccharide components in the extract

[0254] Take 1g of each of the three herbal extracts, add 10ml of hot water, shake thoroughly to dissolve, centrifuge, and use the supernatant to prepare the test solution of the extract according to the direct preparation method described above. A distinct chromatographic peak was found in the Scutellaria baicalensis extract. Mass spectrometry analysis of the corresponding chromatographic peak in the Scutellaria baicalensis extract showed that the molecular weight interval of each fragment ion was 132 (C5H). 10 The presence of O5-H2O suggests that it may be a fragment of a pentose sugar.

[0255] The chromatographic peaks of the dextran standard derivative with a molecular weight of 5000 Da showed multiple molecular weight intervals of 162 m / z (C6H). 12 The fragment peaks (O6-H2O) indicate that this is a hexose sugar, and glucose is the most typical hexose sugar.

[0256] The high-performance liquid chromatograms of the other two herbal extracts did not show obvious high molecular weight peaks. Therefore, the possibility of polysaccharide components in these two extracts can be largely ruled out.

[0257] ② Analysis of polysaccharide components in Tanreqing Injection

[0258] Fifteen batches of Tanreqing injection samples were collected, and test solutions were prepared using the direct method. The chromatograms of the obtained test solutions were compared with the chromatograms of Scutellaria baicalensis extract and dextran standard solutions, respectively.

[0259] The results showed that no obvious chromatographic peaks were observed in Tanreqing injection at the left side of the dextran standard peak (where the molecular weight is larger) or at the position of the polysaccharide peak in Scutellaria baicalensis extract. This indicates that no polysaccharide components with a molecular weight greater than or equal to 5000 Da were detected in the 15 batches of Tanreqing injection samples.

[0260] ③ Spiking recovery and detection limit test

[0261] Accurately measure 1 ml of 10 mg / ml dextran standard solution with a molecular weight of 5000 Da and 10 mg / ml dextran standard solution with a molecular weight of 12000 Da, respectively, for two sample loading levels, and place them in 10 ml volumetric flasks, three copies of each. Dilute to the mark with sample number 1 Tanreqing sample and use as the test sample. Prepare the test sample solution according to the direct preparation steps described above, and calculate the sample recovery rate and detection limit. The results are shown in Tables 3 and 4.

[0262] Table 3. Results of Polysaccharide Recovery Rate Determination in Tanreqing Injection (Direct Method)

[0263]

[0264]

[0265] Table 4. Detection Limits for Polysaccharides in Tanreqing Injection (Direct Method)

[0266] beta-glucan Concentration of the sample solution (μg / ml) Signal-to-noise ratio Detection limit (μg / ml) Calculated using 5000 Da beta 1 51.3 0.06 Calculated using 12000 Da beta 1 47.5 0.06

[0267] 3.2.2.2 Indirect method (acid hydrolysis of polysaccharides + derivatization of monosaccharides)

[0268] ① Analysis of polysaccharide components in the extract

[0269] Take 1g of each of the three herbal extracts, add 10ml of hot water, shake thoroughly to dissolve, centrifuge, and use the supernatant to prepare the test solution of the extracts according to the indirect method described above. Simultaneously, prepare the test solution using the same method with the mixed extract solution provided by the company.

[0270] The results showed that a distinct arabinose chromatographic peak was detected in the Scutellaria baicalensis extract, along with small amounts of galactose and galacturonic acid (based on the localization of each monosaccharide component). The measured arabinose levels were consistent with the conclusions drawn from the previous direct method determination of pentoses. Trace amounts of glucose and arabinose were detected in the Lonicera japonica extract, and trace amounts of arabinose were also detected in the Forsythia suspensa extract. Given that arabinogalactan is a major component of plant polysaccharides, the above results are reasonable.

[0271] Only a small amount of glucose and arabinose were detected in the above intermediate mixture solution. Since the intermediate mixture solution is subjected to activated carbon decolorization and ultrafiltration steps beforehand, the corresponding sugar components should not be detected in the final sample.

[0272] ② Analysis of polysaccharide components in Tanreqing Injection

[0273] Following the indirect preparation method described above, test solutions were prepared for 15 batches of samples for determination by high-performance liquid chromatography (HPLC). The results showed that no polysaccharide components with a molecular weight greater than or equal to 5000 Da were detected in any of the samples.

[0274] ③ Spiking recovery and detection limit test

[0275] Accurately measure 1 ml of 1 mg / ml and 10 mg / ml dextran standard solutions with a molecular weight of 5000 Da, and 1 mg / ml and 10 mg / ml dextran standard solutions with a molecular weight of 12000 Da, for a total of four spiking levels. Place each solution in a 10 ml volumetric flask, making three copies of each. Dilute each sample to the mark with sample number 1 (Tanreqing) to prepare the spiking test solution. Prepare the spiking test solution according to the indirect method described above, and calculate the sample recovery rate and detection limit. The results are shown in Tables 5 and 6.

[0276] Table 5. Results of Polysaccharide Recovery Rate Determination in Tanreqing Injection (Indirect Method)

[0277]

[0278]

[0279] Table 6. Detection Limits for Polysaccharides in Tanreqing Injection (Indirect Method)

[0280] beta-glucan Concentration of the sample solution (μg / ml) Signal-to-noise ratio Detection limit (μg / ml) Calculated using 5000 Da beta 0.1 5.8 0.05 Calculated using 12000 Da beta 0.1 10.2 0.03

[0281] 4. Conclusion

[0282] This paper uses polysaccharide pyridine amination and acid hydrolysis monosaccharide derivatization methods to determine the possible polysaccharide components in Tanreqing injection. These two methods each have their advantages and complement each other, forming a set of specific, sensitive, and efficient detection methods that can effectively control the quality of polysaccharide components. The results show that a small amount of plant polysaccharides are indeed introduced during the preparation of Tanreqing injection. However, these components can be largely removed after decolorization with activated carbon and ultrafiltration in the preparation process. Therefore, no polysaccharide components such as dextran were detected in the Tanreqing injection samples within their effective shelf life.

[0283] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for detecting polysaccharides in Tanreqing injection, characterized in that, The detection method includes the following steps: The test solution and dextran standard solution were prepared and tested using a direct method. The chromatographic conditions for detection in the direct method are as follows: a chromatographic column packed with a mixed gel, a mobile phase of saline solution, a flow rate of 0.1–1.0 ml / min, a column temperature of 10–30 °C, a fluorescence detector, an excitation wavelength of 300–350 nm, and an emission wavelength of 370–430 nm; and The test solution and dextran standard solution were prepared and tested using an indirect method. The chromatographic conditions for detection in the indirect method are as follows: a chromatographic column packed with octadecyl bonded silica gel is used; mobile phase A is selected from one or more of acetonitrile, methanol, and tetrahydrofuran; mobile phase B is an aqueous acid solution, an aqueous alkaline solution, and / or an aqueous buffer solution; the gradient elution program is: 0–2 min, 90% B, 2–30 min, 90→70% B; the flow rate is 0.5–1.5 ml / min; the column temperature is 10–30 °C; and the detection wavelength is 200–400 nm. Based on the detection results of the direct method and the indirect method, it is determined whether the Tanreqing injection contains polysaccharide substances, wherein the polysaccharide substances are glucan.

2. The detection method according to claim 1, characterized in that, The Tanreqing injection is composed of Scutellaria baicalensis, bear bile powder, goat horn, honeysuckle, forsythia, and propylene glycol.

3. The detection method according to claim 1, characterized in that, The steps for preparing the test solution using the direct method include: (a) Take an appropriate amount of Tanreqing injection and purify it through a gel permeation chromatography column to obtain a polysaccharide sample solution; (b) After drying the polysaccharide sample solution, add the coupling reagent solution, mix, and heat to obtain the first solution; (c) Add a reducing agent solution to the first solution, mix, and heat to obtain a second solution; and (d) Add water and phenol-chloroform solution to the second solution, centrifuge, discard the lower organic phase, add water and phenol-chloroform solution again, centrifuge, discard the lower organic phase, add water and phenol-chloroform solution a third time, centrifuge, discard the lower organic phase, and then filter the upper liquid through a membrane to obtain the test solution. Preferably, the steps for preparing the dextran standard solution by the direct method include: weighing an appropriate amount of dextran standard, dissolving it in water to obtain a dextran solution, and then proceeding with the same steps as steps (b) to (d) in the preparation method of the test sample solution to obtain the dextran standard solution.

4. The detection method according to claim 3, characterized in that, Steps (a) to (d) of the direct method for preparing the test solution include any one or more of the following items [1] to [17]: [1] The method of purifying Tanreqing injection in step (a) by gel permeation chromatography includes the following steps: take a pre-packed gel permeation chromatography column, shake it upside down to make the internal packing filler uniformly fill the inside of the pre-packed column, and minimize the generation of air bubbles; cut off the seal at the bottom of the pre-packed column, open the top cap of the pre-packed column, place it in a collection tube, centrifuge for the first time, discard the buffer solution obtained, then place the pre-packed column on another collection tube, add the Tanreqing injection to be purified on the column, centrifuge for the second time, and obtain a polysaccharide sample solution; Preferably, the gel is a polyacrylamide gel; Preferably, the gel permeation chromatography pre-packed column is a Bio-Spin 6Tris column; More preferably, the centrifugation speed is 2000-4000 r / min, for example, about 3000 r / min; Preferably, the first centrifugation time is 1 to 5 minutes, for example, about 2 minutes; More preferably, the second centrifugation time is 1 to 10 minutes, for example, about 4 minutes; Particularly preferably, the volume of the Tanreqing injection is 50–150 μl, for example, about 100 μl; Particularly preferably, the volume of the polysaccharide sample solution is 50–150 μl, for example, about 100 μl; [2] The drying process in step (b) is nitrogen drying; [3] The method for preparing the coupling reagent solution in step (b) is to dissolve 2-aminopyridine in glacial acetic acid to prepare a glacial acetic acid solution of 2500-3000 mg / ml, for example, about 2760 mg / ml of 2-aminopyridine. [4] The volume ratio of the polysaccharide sample solution in step (a) to the coupling reagent solution in step (b) is (1 to 10):1, for example, about 5:1; [5] The heating temperature in step (b) is 85°C to 95°C, for example, about 90°C; [6] The heating time in step (b) is 50 min to 70 min, for example, about 60 min; [7] The method for preparing the reducing agent solution in step (c) is to dissolve the boric acid 200-dimethylamine complex in acetic acid and water to prepare an acetic acid aqueous solution of boric acid 200-dimethylamine complex with a concentration of 1200-2000 mg / ml, for example, about 1538 mg / ml, wherein the volume ratio between acetic acid and water is about 8: about 5. [8] The volume ratio of the polysaccharide sample solution in step (a) to the reducing agent solution in step (c) is (5-12):7, for example, about 10:7; [9] The heating temperature in step (c) is 75°C to 85°C, for example, about 80°C; [10] The heating time in step (c) is 25 to 45 minutes, for example, about 35 minutes; [11] The volume ratio of the polysaccharide sample solution in step (a) to the water in step (d) is (5-12):9, for example, about 10:9; [12] The volume ratio of the phenol-chloroform solution in step (d) is (0.1-2):(0.1-2), for example, about 1:about 1; [13] The volume ratio of the polysaccharide sample solution in step (a) to the phenol-chloroform solution in step (d) is (5-12):9, for example, about 10:9; [14] The volume ratio of the water to the phenol-chloroform solution in step (d) is (0.1-2):(0.1-2), for example, about 1:about 1; [15] The centrifugation speed in step (d) is 5000 to 7000 r / min, for example about 6000 r / min; [16] The centrifugation time in step (d) is 30 to 90 seconds, for example, about 60 seconds; [17] The pore size of the filter membrane in step (d) is 0.45 μm.

5. The detection method according to claim 1, characterized in that, The steps for preparing the test solution using the indirect method include: (A) Take an appropriate amount of Tanreqing injection and purify it through a gel permeation chromatography column to obtain a polysaccharide sample solution; (B) Add trifluoroacetic acid solution to the polysaccharide sample solution, vortex, hydrolyze, cool, and dry to obtain the first sample; (C) Water is added to the first sample to make up the total volume, then ammonia solution and PMP methanol solution are added, vortexed, reacted, cooled, and dried to obtain the second sample; and (D) Add KH2PO4 solution and chloroform to the second sample, vortex, let stand, discard the lower organic phase, add chloroform again, vortex, let stand, discard the lower organic phase, add chloroform a third time, vortex, let stand, discard the lower organic phase, and then filter the upper liquid through a membrane to obtain the test solution. Preferably, the indirect method for preparing the dextran standard solution includes: weighing an appropriate amount of dextran standard, dissolving it in water to obtain a dextran solution, and then proceeding with the same steps (C) to (D) as in the preparation method of the test sample solution to obtain the dextran standard solution.

6. The detection method according to claim 5, characterized in that, Steps (A) to (D) of the indirect method for preparing the test solution include any one or more of the following items [1] to [21]: [1] The method of purifying Tanreqing injection in step (A) by gel permeation chromatography includes the following steps: take a pre-packed gel permeation chromatography column, shake it upside down to make the internal packing filler uniformly fill the inside of the pre-packed column, and minimize the generation of air bubbles; cut off the seal at the bottom of the pre-packed column, open the top cap of the pre-packed column, place it in a collection tube, centrifuge for the first time, discard the buffer solution obtained, then place the pre-packed column on another collection tube, add the Tanreqing injection to be purified on the column, centrifuge for the second time, and obtain a polysaccharide sample solution; Preferably, the gel is a polyacrylamide gel; Preferably, the gel permeation chromatography pre-packed column is a Bio-Spin 6Tris column; More preferably, the centrifugation speed is 2000-4000 r / min, for example, about 3000 r / min; Preferably, the first centrifugation time is 1 to 5 minutes, for example, about 2 minutes; More preferably, the second centrifugation time is 1 to 10 minutes, for example, about 4 minutes; Particularly preferably, the volume of the Tanreqing injection is 50–150 μl, for example, about 100 μl; Particularly preferably, the volume of the polysaccharide sample solution is 50–150 μl, for example, about 100 μl; [2] The molar concentration of trifluoroacetic acid in step (B) is 2 to 6 mol / L, for example, about 4 mol / L; [3] The volume ratio of the polysaccharide sample solution in step (A) to the trifluoroacetic acid in step (B) is (0.5 to 5):1, for example, about 1:1; [4] The vortex in step (B) lasts for 10 to 60 seconds, for example, about 30 seconds; [5] The hydrolysis temperature in step (B) is 90 to 130°C, for example, about 110°C; [6] The hydrolysis time in step (B) is 30 to 90 minutes, for example, about 60 minutes; [7] The drying process described in step (B) or step (C) is nitrogen drying; [8] The volume ratio of the polysaccharide sample solution in step (A) to the total amount of water added in step (C) is (0.5 to 5):1, for example, about 1:1; [9] The volume percentage concentration of the ammonia solution in step (C) is 10% to 30%, for example, about 20%; [10] The volume ratio of the polysaccharide sample solution in step (A) to the ammonia solution in step (C) is (0.5 to 5):1, for example, about 1:1; [11] The molar concentration of the PMP methanol solution in step (C) is 0.2 to 0.8 mol / L, for example, about 0.5 mol / L; [12] The volume ratio of the polysaccharide sample solution in step (A) to the PMP methanol solution in step (C) is (0.5 to 5):1, for example, about 1:1; [13] The vortex in step (C) lasts for 10 to 60 seconds, for example, about 30 seconds; [14] The temperature of the reaction in step (C) is 50 to 90°C, for example about 70°C; [15] The reaction time in step (C) is 30 to 90 minutes, for example, about 60 minutes; [16] The molar concentration of the KH2PO4 solution in step (D) is 0.01 to 0.2 mol / L, for example, about 0.1 mol / L; [17] The volume ratio of the polysaccharide sample solution in step (A) to the KH2PO4 solution in step (D) is 1:(15-25), for example, 1:about 20; [18] The volume ratio of the polysaccharide sample solution in step (A) to the chloroform in step (D) is 1:(15-25), for example, 1:about 20; [19] The vortex in step (D) lasts for 10 to 60 seconds, for example, about 30 seconds; [20] The settling time in step (D) is 2 to 8 minutes, for example, about 5 minutes; [21] The pore size of the filter membrane in step (D) is 0.45 μm.

7. The detection method according to claim 1, characterized in that, The acid aqueous solution, alkaline aqueous solution and / or buffer salt aqueous solution are selected from one or more weak acids and their salts, weak bases and their salts of different concentrations; Preferably, the acidic aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution are selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid; More preferably, the acid aqueous solution is a 0.005% to 0.12% acid aqueous solution; Preferably, the buffer salt solution is a phosphate solution and / or an acetate solution; Preferably, the pH value of the buffer salt solution is not greater than 7.2, for example, about 6.8; Particularly preferred is that the detection wavelength is 220–280 nm, for example, 250 nm; Particularly preferred, the peak purity of the chromatographic peak corresponding to the reference standard is greater than 995; Particularly preferred is that the resolution of the chromatographic peak corresponding to the reference standard is greater than 2.

0.

8. The detection method according to claim 1, characterized in that, The exclusion limit of the hybrid gel is 7 × 10⁻⁶. 6 ; Preferably, the polysaccharide detected in the Tanreqing injection is a plant polysaccharide with a molecular weight greater than or equal to 5000 Da; More preferably, the salt solution is an aqueous solution of sodium nitrate; Preferably, the molar concentration of the sodium nitrate aqueous solution is 0.01–1.0 mol / L, for example, about 0.1 mol / L; More preferably, the flow rate is 0.2 to 0.6 ml / min, for example, about 0.4 ml / min; Particularly preferred is that the excitation wavelength is 320–350 nm, for example, about 330 nm; Particularly preferred is that the emission wavelength is 375–425 nm, for example, about 400 nm.

9. The detection method according to claim 1, characterized in that, The criteria for identifying whether the Tanreqing injection contains polysaccharides include: comparing the chromatogram of the test solution with the chromatogram of the polysaccharide standard solution to check whether there is a corresponding chromatographic peak at the position of the polysaccharide chromatographic peak in the test solution; if there is no corresponding chromatographic peak in both the direct method and the indirect method, then the Tanreqing injection does not contain polysaccharides, wherein the polysaccharide is dextran.

10. Use of the detection method according to any one of claims 1 to 9 in the quality detection and / or quality evaluation and / or quality control of polysaccharides in Tanreqing Injection.

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