A method for constructing a fingerprint spectrum of ganoderma lucidum oral liquid and a method for detecting ganoderma lucidum polysaccharide content

Abstract

The present application relates to the technical field of analysis and detection, in particular to a method for constructing a fingerprint spectrum of Ganoderma lucidum oral liquid and a method for detecting Ganoderma lucidum polysaccharide content. The fingerprint spectrum constructed by the present application contains two types of effective active ingredients, i.e. terpenes (such as ganoderic acid A) and Ganoderma lucidum polysaccharide, and can comprehensively, accurately and comprehensively reflect the overall quality of the Ganoderma lucidum oral liquid, thereby providing a comprehensive identification and evaluation method for quality control of the Ganoderma lucidum oral liquid, so as to better ensure the quality stability, consistency and controllability of the preparation, and provide technical support for improving the intrinsic efficacy of the Ganoderma lucidum oral liquid. The method for detecting Ganoderma lucidum polysaccharide content provided by the present application has good repeatability.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, specifically to a method for constructing a fingerprint spectrum of Ganoderma lucidum oral liquid and a method for detecting the content of Ganoderma lucidum polysaccharides. Background Technology

[0002] Reishi oral liquid has the effects of strengthening the spleen and stomach, calming the mind and soothing the nerves. It is used for neurasthenia caused by deficiency of both heart and spleen, such as insomnia, forgetfulness, lethargy, and fatigue. Due to its good medicinal value, it has been widely used, therefore, its quality control needs to be strengthened. Studies have shown that the main effective components of Reishi include polysaccharides, terpenes, sterols, and alkaloids, which have various pharmacological effects such as immunomodulation, anti-tumor, anticonvulsant, and lipid-lowering. Therefore, the detection of polysaccharides and terpenes in Reishi oral liquid is of important pharmaceutical significance.

[0003] Gel permeation chromatography (GPC / SEC) is a commonly used technique for analyzing the molecular weight and distribution of polysaccharides, and it is now widely used in the quality control of various plant and fungal polysaccharides. High-performance liquid chromatography (HPLC) is a commonly used technique for analyzing ganoderic acid A.

[0004] However, as a compound preparation, Ganoderma lucidum oral liquid has a complex matrix composition. Besides the target polysaccharide and ganoderic acid A, it also contains various other interfering substances such as terpenes. Directly applying existing gel chromatography analysis methods for plant polysaccharides and HPLC detection methods for ganoderic acid A often results in problems such as poor peak resolution, severe baseline drift, and poor retention time repeatability, making it difficult to obtain stable and reproducible polysaccharide characteristic chromatograms. For example, related technologies disclose methods for preparing and analyzing the quality of standard decoctions of Ganoderma lucidum slices, with the following chromatographic conditions: Column: Thermo-C 18 The chromatographic column was 4.6 mm × 250 mm, 5 μm; the mobile phase was acetonitrile (A) - 0.1% acetic acid (B), with gradient elution (0–20 min, 72%–68% B; 20–40 min, 68%–64% B); the flow rate was 1.0 mL / min; the column temperature was 30 °C; the injection volume was 10 μL; the detection wavelength was 256 nm; the theoretical plate number, calculated based on ganoderic acid A, should be no less than 3000. However, under these chromatographic conditions, ganoderic acid A did not produce a peak in the Ganoderma lucidum oral liquid.

[0005] Therefore, there is an urgent need to establish a fingerprinting method suitable for Ganoderma lucidum oral liquids to achieve overall characterization and effective identification of its polysaccharide and terpene components (such as ganoderic acid A), and to provide a reliable technical means for the quality control of this type of oral liquid. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a method for constructing a fingerprint spectrum of Ganoderma lucidum oral liquid. The fingerprint spectrum constructed in this invention can effectively reflect the overall quality of Ganoderma lucidum oral liquid and provide effective identification.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for constructing a fingerprint spectrum of Ganoderma lucidum oral liquid, wherein the fingerprint spectrum includes a terpene fingerprint spectrum and a Ganoderma lucidum polysaccharide fingerprint spectrum; The method for constructing the terpene fingerprint includes the following steps: The Ganoderma lucidum oral liquid was diluted with water to obtain a terpenoid test solution. The terpene test solution and the ganoderic acid A reference solution were respectively subjected to high performance liquid chromatography to obtain the liquid chromatograms of the test sample and the reference sample, respectively. The conditions for high-performance liquid chromatography (HPLC) detection include: a C18 column; a mobile phase system comprising mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is a 0.8-0.12% (v / v) formic acid aqueous solution; and a gradient elution method, wherein the gradient elution procedure is as follows: From 0 to 25 minutes, the volume fraction of the mobile phase A increased from 27-28.5% to 29-32%. Over 25-50 minutes, the volume fraction of mobile phase A increased from 29-32% to 37-41%. Over 50-60 minutes, the volume fraction of mobile phase A increased from 37-41% to 58-62%. Over 60-70 minutes, the volume fraction of mobile phase A decreased from 58-62% to 27-28.5%. The retention times of the liquid chromatography chromatogram of the test sample and the liquid chromatography chromatogram of the reference sample were compared to obtain the terpene fingerprint chromatogram. The method for constructing the Ganoderma lucidum polysaccharide fingerprint spectrum includes the following steps: Ganoderma lucidum oral liquid was dissolved in hot water and precipitated with ethanol to obtain crude Ganoderma lucidum polysaccharide; the crude Ganoderma lucidum polysaccharide was dissolved in sodium chloride aqueous solution with a concentration of 0.1~0.3mol / L to obtain Ganoderma lucidum polysaccharide test solution; The fingerprint spectrum and the Ganoderma lucidum polysaccharide reference solution were subjected to gel chromatography to obtain gel chromatograms of the test sample and the reference sample, respectively; the mobile phase for gel chromatography included an aqueous sodium chloride solution with a concentration of 0.1~0.3 mol / L. The molecular weight of the test sample gel chromatography was compared with that of the reference sample gel chromatography to obtain the Ganoderma lucidum polysaccharide fingerprint.

[0008] Preferably, the conditions for the high-performance liquid chromatography detection further include: a Zafex C10 column. 18 The column temperature is 25~35℃; the mobile phase flow rate is 0.8~1.2mL / min; the injection volume is 5~15μL; and the detection wavelength is 256nm.

[0009] Preferably, the volume ratio of the Ganoderma lucidum oral liquid to the diluting water is 1:0.5~1.5; The dilution process also includes centrifuging the diluted solution.

[0010] Preferably, the concentration of ganoderic acid A reference standard in the ganoderic acid A reference solution is 0.2~0.5 mg / mL, and the solvent includes methanol.

[0011] Preferably, the conditions for gel chromatography detection further include: a Monomix MC10-SEC column; a detection cell temperature of 35~45℃; a column temperature of 35~45℃; a mobile phase flow rate of 0.3~0.7mL / min; and an injection volume of 20~80μL.

[0012] Preferably, the ratio of the Ganoderma lucidum oral liquid to hot water is 1g:10~30mL; The ratio of the amount of Ganoderma lucidum oral liquid to the amount of ethanol used for precipitation is 1g: 200~400mL; The precipitation was carried out at a temperature of 2-6°C for 8-16 hours. The ratio of the amount of Ganoderma lucidum oral liquid to the sodium chloride aqueous solution for dissolving is 1g:30~70mL.

[0013] The precipitation process further includes: sequentially centrifuging the precipitated system, and washing the resulting solid components with ethanol to remove the ethanol.

[0014] Preferably, the Ganoderma lucidum polysaccharide reference standard in the Ganoderma lucidum polysaccharide reference standard solution includes a set of dextran molecular weight standards, which includes dextran with molecular weights of 180, 2700, 5250, 9750, 13050 and 36800 respectively. The solvent in the Ganoderma lucidum polysaccharide reference solution includes an aqueous sodium chloride solution with a concentration of 0.1~0.3 mol / L.

[0015] This invention also provides a method for detecting the content of Ganoderma lucidum polysaccharides in Ganoderma lucidum oral liquid, comprising the following steps: Ganoderma lucidum oral liquid and ethanol are mixed and precipitated to obtain a precipitate. The precipitate is then dissolved in water to obtain a test solution. The precipitation is carried out 3 to 5 times. The absorbance of the test solution was tested, and the test results of the Ganoderma lucidum polysaccharide content in the Ganoderma lucidum oral liquid were obtained according to the absorbance and the absorbance-concentration standard curve; the absorbance-concentration standard curve used the glucose concentration as the independent variable and the absorbance as the dependent variable.

[0016] Preferably, the precipitation temperature is 2~8℃, and the precipitation time for a single precipitation is 30~60min; The dissolution is carried out under ultrasonic conditions, and the dissolution time is 15-30 minutes. The absorbance testing methods include the phenol-sulfuric acid method or the sulfuric acid-anthrone method.

[0017] The present invention also provides the application of the method for constructing the fingerprint spectrum of Ganoderma lucidum oral liquid described in the above technical solution or the method for detecting the content of Ganoderma lucidum polysaccharides in Ganoderma lucidum oral liquid described in the above technical solution in the quality control of Ganoderma lucidum oral liquid.

[0018] The fingerprint spectrum constructed in this invention contains two types of effective active ingredients: terpenoids (such as ganoderic acid A) and Ganoderma lucidum polysaccharides. It can comprehensively, accurately, and holistically reflect the overall quality of Ganoderma lucidum oral liquid, providing a comprehensive identification and evaluation method for the quality control of Ganoderma lucidum oral liquid. This better ensures the quality stability, consistency, and controllability of Ganoderma lucidum oral liquid, and provides technical support for improving the intrinsic efficacy of Ganoderma lucidum oral liquid.

[0019] Existing methods for detecting Ganoderma lucidum polysaccharide content in Ganoderma lucidum oral liquids exhibit poor reproducibility. The detection method provided by this invention offers stable colorimetric results and accurate quantification. Furthermore, the method demonstrates good repeatability, enabling accurate and highly sensitive detection of Ganoderma lucidum polysaccharide content in oral liquids. This provides a comprehensive identification and evaluation method for the quality control of Ganoderma lucidum oral liquids, thereby better ensuring the quality stability, consistency, and controllability of the oral liquids and providing technical support for enhancing their intrinsic efficacy. Attached Figure Description

[0020] Figure 1 The HPLC chromatogram of the ganoderic acid A reference solution in Comparative Example 1 is shown. Figure 2 The HPLC chromatogram of the terpenoid test solution in Comparative Example 1 is shown. Figure 3 The HPLC chromatogram of the ganoderic acid A reference solution in Comparative Example 2 is shown. Figure 4 The HPLC chromatogram of the ganoderic acid A reference solution in Comparative Example 3 is shown. Figure 5 The HPLC chromatogram of the ganoderic acid A reference solution in Comparative Example 4 is shown. Figure 6 The HPLC chromatogram of the ganoderic acid A reference solution in Example 1 is shown below. Figure 7 The spectrum is of the terpenoid test solution prepared from the Ganoderma lucidum oral liquid in Example 1; Figure 8 The above are superimposed HPLC chromatograms of the test solutions prepared from the three batches of Ganoderma lucidum oral liquid in Example 2; Figure 9 This is a comparison of the superimposed HPLC chromatograms of the three batches of Ganoderma lucidum oral liquid and the chromatogram of the extract of Ganoderma lucidum oral liquid in Example 2; Figure 10 This is a control chromatogram generated from three batches of Ganoderma lucidum oral liquid in Example 2; Figure 11 This is a gel chromatogram of the Ganoderma lucidum polysaccharide test solution in Example 3; Figure 12 The above are comparative gel chromatograms of the extract of Ganoderma lucidum oral liquid and three batches of Ganoderma lucidum polysaccharide test solutions in Example 3. Figure 13 This is a control chromatogram generated from the extract of Ganoderma lucidum oral liquid and three batches of Ganoderma lucidum polysaccharide test solutions in Example 3; Figure 14 This is the standard curve in Example 5; Figure 15 The standard curve is shown in Comparative Example 6; Figure 16 This refers to the standard curve in Example 7; Figure 17 This is the standard curve in Example 8. Detailed Implementation

[0021] This invention provides a method for constructing a fingerprint spectrum of Ganoderma lucidum oral liquid, wherein the fingerprint spectrum includes a terpene fingerprint spectrum and a Ganoderma lucidum polysaccharide fingerprint spectrum; The method for constructing the terpene fingerprint includes the following steps: The Ganoderma lucidum oral liquid was diluted with water to obtain a terpenoid test solution. The terpene test solution and the ganoderic acid A reference solution were respectively subjected to high performance liquid chromatography to obtain the liquid chromatograms of the test sample and the reference sample, respectively. The conditions for the high-performance liquid chromatography (HPLC) detection include: a C18 column; a mobile phase system comprising mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is a 0.9-0.11% (v / v) formic acid aqueous solution; and a gradient elution method, wherein the gradient elution procedure is as follows: From 0 to 25 minutes, the volume fraction of the mobile phase A increased from 27-28.5% to 29-32%. Over 25-50 minutes, the volume fraction of mobile phase A increased from 29-32% to 37-41%. Over 50-60 minutes, the volume fraction of mobile phase A increased from 37-41% to 58-62%. Over 60-70 minutes, the volume fraction of mobile phase A decreased from 58-62% to 27-28.5%. The retention times of the liquid chromatography chromatogram of the test sample and the liquid chromatography chromatogram of the reference sample were compared to obtain the terpene fingerprint chromatogram. The method for constructing the Ganoderma lucidum polysaccharide fingerprint spectrum includes the following steps: Ganoderma lucidum oral liquid was dissolved in hot water and precipitated with ethanol to obtain crude Ganoderma lucidum polysaccharide; the crude Ganoderma lucidum polysaccharide was dissolved in sodium chloride aqueous solution with a concentration of 0.1~0.3mol / L to obtain Ganoderma lucidum polysaccharide test solution; The fingerprint spectrum and the Ganoderma lucidum polysaccharide reference solution were subjected to gel chromatography to obtain gel chromatograms of the test sample and the reference sample, respectively; the mobile phase for gel chromatography included an aqueous sodium chloride solution with a concentration of 0.1~0.3 mol / L. The molecular weight of the test sample gel chromatography was compared with that of the reference sample gel chromatography to obtain the Ganoderma lucidum polysaccharide fingerprint.

[0022] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0023] The method for constructing terpene fingerprints will be explained below.

[0024] This invention involves diluting Ganoderma lucidum oral liquid with water to obtain a terpenoid test solution. In this invention, the volume ratio of the Ganoderma lucidum oral liquid to the diluting water can be 1:0.5~1.5, or 1:0.8~1.2, or even 1:0.9~1.1, specifically 1:1. In this invention, the dilution process further includes centrifuging the diluted solution, with the supernatant being the terpenoid test solution. In this invention, the centrifugation speed can be 8000~12000 r / min, or 9000~11000 r / min, specifically 10000 r / min; the centrifugation time can be 5~15 min, or 8~12 min, specifically 10 min.

[0025] After obtaining the terpenoid test solution, the present invention performs high performance liquid chromatography on the terpenoid test solution and the ganoderic acid A reference solution to obtain the test sample liquid chromatogram and the reference sample liquid chromatogram, respectively.

[0026] In this invention, the concentration of ganoderic acid A reference standard in the ganoderic acid A reference standard solution can be 0.2~0.5 mg / mL, or 0.3~0.4 mg / mL, or even 0.35~0.4 mg / mL, specifically 0.384 mg / mL; the solvent in the ganoderic acid A reference standard solution can include methanol.

[0027] In this invention, the conditions for high-performance liquid chromatography (HPLC) detection include: the chromatographic column is a C18 column, specifically a Zafex C18 column. 18 The column temperature can be 25~35℃, or 28~32℃, specifically 30℃; the mobile phase system includes mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile, and mobile phase B is an aqueous formic acid solution with a volume concentration of 0.8~0.12% (can be 0.9~0.11%, specifically 0.1%); the mobile phase flow rate can be 0.8~1.2mL / min, or 0.9~1.1mL / min, specifically 1mL / min; the injection volume can be... The volume of mobile phase A can be 5-15 μL, or 8-12 μL, specifically 10 μL; the detection wavelength can be 256 nm; the elution method is gradient elution, and the gradient elution procedure is as follows: 0-25 min, the volume fraction of mobile phase A increases from 27-28.5% (which can be 27.5-28.4%, or 27.8-28.2%, specifically 28%) to 29-32% (which can be 29.5-31.5%, or 29.8-31%). (Specifically, it can be 30%); 25-50 min, the volume fraction of the mobile phase A increases from 29-32% (which can be 29.5-31.5%, or 29.8-31%, specifically 30%) to 37-41% (which can be 38-40%, or 38.5-40.5%, specifically 40%); 50-60 min, the volume fraction of the mobile phase A increases from 37-41% (which can be 38-40%, or 38.5-40%) to 37-41% (which can be 38-40%, or 38.5-40%). The volume fraction of the mobile phase A increases from 58-62% (which can be 59-61%, 59.5-60.5%, or 60%) to 27-28.5% (which can be 27.5-28.4%, 27.8-28.2%, or 28%) over 60-70 minutes.

[0028] After obtaining the liquid chromatography chromatograms of the test sample and the reference sample, this invention compares the retention times of the two chromatograms to obtain a terpene fingerprint chromatogram. In this invention, a peak appears at the same position as the main component elution peak in the reference sample chromatogram. Simultaneously, the liquid chromatography chromatograms of the test sample and the reference sample are imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)" and compared with the reference chromatogram; the similarity is greater than 0.9.

[0029] The method for constructing the fingerprint spectrum of Ganoderma lucidum polysaccharides is described below.

[0030] In this invention, Ganoderma lucidum oral liquid is dissolved in hot water and precipitated with ethanol to obtain crude Ganoderma lucidum polysaccharide; the crude Ganoderma lucidum polysaccharide is then dissolved in an aqueous sodium chloride solution with a concentration of 0.1~0.3mol / L to obtain a Ganoderma lucidum polysaccharide test solution.

[0031] In this invention, the temperature of the hot water can be 95~100℃. In this invention, the ratio of the amount of Ganoderma lucidum oral liquid to the amount of hot water can be 1g:10~30mL, or 1g:15~25mL, or even 1g:18~22mL, or specifically 1g:20mL.

[0032] In this invention, the ratio of the Ganoderma lucidum oral liquid to the ethanol used for precipitation can be 1g:200-400mL, or 1g:250-350mL, or even 1g:280-320mL, specifically 1g:300mL. In this invention, the precipitation temperature can be 2-6℃, or 3-5℃, specifically 4℃; the precipitation time can be 8-16h, or 10-14h, or even 11-13h, specifically 12h.

[0033] In this invention, the precipitation process may further include: sequentially centrifuging the precipitated system, and washing the resulting solid components with ethanol to remove ethanol. In this invention, the centrifugation speed can be 2000-6000 r / min, or 3000-5000 r / min, specifically 4000 r / min; the centrifugation time can be 5-15 min, or 8-12 min, specifically 10 min. In this invention, the ethanol washing can be performed 1-4 times, or 2-3 times. In this invention, the ethanol removal temperature can be 60-100℃, or 70-90℃, specifically 80℃; the ethanol removal may include ethanol removal through evaporation.

[0034] In this invention, the ratio of the Ganoderma lucidum oral liquid to the sodium chloride aqueous solution for dissolving it can be 1g:30-70mL, or 1g:40-60mL, or even 1g:45-55mL, specifically 1g:50mL. In this invention, the concentration of the sodium chloride aqueous solution is 0.1-0.3mol / L, specifically 0.15-0.25mol / L, specifically 0.2mol / L.

[0035] In this invention, after dissolving the crude Ganoderma lucidum polysaccharide in the sodium chloride aqueous solution, the process further includes shaking and filtering, with the filtrate being a Ganoderma lucidum polysaccharide test solution. In this invention, the pore size of the filter membrane used for filtration can be 0.45 μm.

[0036] After obtaining the Ganoderma lucidum polysaccharide test solution, the present invention performs gel chromatography detection on the fingerprint spectrum and the Ganoderma lucidum polysaccharide reference solution respectively to obtain the gel spectrum of the test sample and the gel spectrum of the reference sample respectively; the mobile phase for gel chromatography detection includes an aqueous sodium chloride solution with a concentration of 0.1~0.3mol / L.

[0037] In this invention, the Ganoderma lucidum polysaccharide reference standard solution may include a set of dextran molecular weight standards, which may include dextran with molecular weights of 180, 2700, 5250, 9750, 13050, and 36800; the set of dextran molecular weight standards may also include dextran with molecular weights of 64650, 135350, 300600, and 2000000. In this invention, the solvent in the Ganoderma lucidum polysaccharide reference standard solution may include a sodium chloride aqueous solution with a concentration of 0.1~0.3 mol / L, and the concentration of the sodium chloride aqueous solution may also be 0.15~0.25 mol / L, specifically 0.2 mol / L. This invention obtains a standard curve of Ganoderma lucidum polysaccharides based on the gel permeation chromatography of the reference standard, using the retention time of each dextran as the independent variable and the corresponding molecular weight as the dependent variable.

[0038] In this invention, the conditions for gel chromatography detection include: the chromatographic column can be a Monomix MC10-SEC column, or a Monomix MC10-SEC (7.8 mm × 300 mm, 10 μm, 1000 Å) and Monomix... The MC10-SEC (7.8 mm × 300 mm, 10 μm, 300 Å) tandem chromatographic column is used. The mobile phase is an aqueous sodium chloride solution with a concentration of 0.1–0.3 mol / L, which can be 0.15–0.25 mol / L, or specifically 0.2 mol / L. The detection cell temperature can be 35–45 °C, which can be 38–42 °C, or specifically 40 °C. The column temperature can be 35–45 °C, which can be 38–42 °C, or specifically 40 °C. The mobile phase flow rate can be 0.3–0.7 mL / min, which can be 0.4–0.6 mL / min, or specifically 0.5 mL / min. The injection volume can be 20–80 μL, which can be 30–70 μL, further 40–60 μL, or specifically 50 μL.

[0039] After obtaining the gel chromatography of the test sample and the reference sample, the present invention compares the molecular weights of the gel chromatography of the test sample and the reference sample to obtain the Ganoderma lucidum polysaccharide fingerprint spectrum.

[0040] In this invention, the retention time of Ganoderma lucidum polysaccharides in the gel chromatography of the sample is substituted into the standard curve of the Ganoderma lucidum polysaccharides to obtain the molecular weight of Ganoderma lucidum polysaccharides in the oral liquid.

[0041] This invention imports the gel chromatogram of the test sample and the gel chromatogram of Ganoderma lucidum oral liquid extract into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". The similarity between the gel chromatogram of the test sample and the Ganoderma lucidum oral liquid extract is greater than 0.9.

[0042] This invention also provides a method for detecting the content of Ganoderma lucidum polysaccharides in Ganoderma lucidum oral liquid, comprising the following steps: Ganoderma lucidum oral liquid and ethanol were mixed and precipitated to obtain a precipitate. The precipitate was then dissolved in water to obtain a test solution. The precipitation was performed three times. The absorbance of the test solution was tested, and the test results of the Ganoderma lucidum polysaccharide content in the Ganoderma lucidum oral liquid were obtained according to the absorbance and the absorbance-concentration standard curve; the absorbance-concentration standard curve used the glucose concentration as the independent variable and the absorbance as the dependent variable.

[0043] This invention involves mixing Ganoderma lucidum oral liquid with ethanol, precipitating the mixture to obtain a precipitate, and then dissolving the precipitate in water to obtain a test solution.

[0044] In this invention, the volume ratio of the Ganoderma lucidum oral liquid to the ethanol used for single precipitation can be 1:40~150, or 1:50~100, specifically 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100.

[0045] In this invention, the mixing may include vortex mixing; the vortex mixing lasts for 1 to 2 minutes.

[0046] In this invention, the precipitation is performed 3 to 5 times, specifically 3 times; the precipitation temperature is 2 to 8°C, or 3 to 7°C, or even 4 to 6°C, specifically 5°C; the precipitation time for each precipitation is 30 to 60 minutes, specifically 30 minutes, 40 minutes, or 50 minutes.

[0047] In this invention, after each precipitation, the system is further centrifuged; the centrifugation speed can be 3000~5000 r / min, or 3200~4000 r / min, specifically 3500 r / min; the centrifugation time can be 3~7 min, or 4~6 min, specifically 5 min; after the final centrifugation, the obtained solid component is dried to obtain a precipitate; the drying temperature can be 70~90℃, or 75~85℃, specifically 80℃; this invention does not have a special limitation on the drying time, as long as the solvent is evaporated.

[0048] In this invention, the dissolution can be carried out under ultrasonic conditions, and the dissolution time can be 15-30 min, or 20-25 min, specifically 20 min; the dissolution temperature can be 20-30℃, specifically room temperature; the ultrasonic power can be 60-100W, or 70-90W, specifically 80W.

[0049] In this invention, the volume ratio of the Ganoderma lucidum oral liquid to the water for dissolving can be 1:10~50, or 1:20~40, or specifically 1:25, 1:30 or 1:35.

[0050] After obtaining the test solution, the present invention tests the absorbance of the test solution, and obtains the test results of the Ganoderma lucidum polysaccharide content in the Ganoderma lucidum oral liquid based on the absorbance and the absorbance-concentration standard curve; the absorbance-concentration standard curve uses the concentration of glucose as the independent variable and the absorbance as the dependent variable.

[0051] In this invention, the absorbance testing method includes the phenol-sulfuric acid method or the sulfuric acid-anthrone method.

[0052] In this invention, the method for testing absorbance using the phenol-sulfuric acid method may include the following steps: mixing the test solution, an aqueous phenol solution, and sulfuric acid, performing a colorimetric reaction to obtain a test solution, and testing the absorbance of the test solution at 490 nm to obtain the sample absorbance A. 样品 Using the corresponding colorimetric reagents (phenol aqueous solution and sulfuric acid) as blank solutions, the absorbance of the blank solutions at 490 nm was measured to obtain the blank absorbance A. 空白 The formula for calculating absorbance ΔA is as follows: ΔA = A 样品 -A 空白 .

[0053] In this invention, the mass-volume concentration of the phenol aqueous solution can be 2-6%, or 3-5%, specifically 4%. In this invention, the volume ratio of the test solution to the phenol aqueous solution can be 1:1 to 1:3, specifically 1:2.

[0054] In this invention, the ratio of the test solution to sulfuric acid can be 0.2 mL: 65-67 g, or 0.2 mL: 65.5-66.5 g, specifically 0.2 mL: 66 g. In this invention, the mass concentration of the sulfuric acid is 95-100%, or 96-99%, specifically 98%.

[0055] In this invention, the method for testing absorbance using the sulfuric acid anthrone method may include the following steps: mixing the test solution and the sulfuric acid anthrone solution, performing a colorimetric reaction to obtain the test solution, and testing the absorbance of the test solution at 490 nm to obtain the sample absorbance A. 样品 Using the corresponding colorimetric reagent (anthrone sulfate solution) as a blank solution, the absorbance of the blank solution at 490 nm was measured to obtain the blank absorbance A. 空白 The formula for calculating absorbance ΔA is as follows: ΔA = A 样品 -A 空白 .

[0056] In this invention, the anthrone sulfate solution is obtained by dissolving anthrone in sulfuric acid. In this invention, the concentration of anthrone in the anthrone sulfate solution can be 0.5~1.5 g / L, or 0.8~1.2 g / L, specifically 1 g / L. In this invention, the mass concentration of the sulfuric acid can be 95~100%, or 96~99%, specifically 98%.

[0057] In this invention, the volume ratio of the test solution to the anthrone sulfate solution can be 1:2 to 4, or 1:2.5 to 3.5, and specifically 1:3.

[0058] In this invention, the method for plotting the absorbance-concentration standard curve may include the following steps: preparing a series of glucose reference solutions of different concentrations; testing the absorbance of the glucose reference solutions of different concentrations to obtain the absorbance of the standard; plotting a standard curve with the concentration of the glucose reference solutions of different concentrations as the independent variable and the absorbance of the standard at the corresponding concentration as the dependent variable to obtain the absorbance-concentration standard curve.

[0059] In this invention, the preparation method of the series of glucose reference solutions includes the following steps: mixing anhydrous glucose standard and water to obtain a glucose standard stock solution; diluting the glucose standard stock solution with water as a solvent to obtain glucose reference solutions of a series of concentrations. In this invention, the concentration of the glucose standard stock solution can be 0.1~0.2 mg / mL, or 0.1~0.15 mg / mL, specifically 0.1 mg / mL. In this invention, the series of glucose reference solutions are obtained by precisely adding water to 5.0 mL of 0.0 mL, 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL of glucose standard stock solution, respectively.

[0060] In this invention, the method for testing the absorbance of the standard is the same as the method for testing the absorbance of the test solution.

[0061] The present invention also provides the application of the method for constructing the fingerprint spectrum of Ganoderma lucidum oral liquid described in the above technical solution or the method for detecting the content of Ganoderma lucidum polysaccharides in Ganoderma lucidum oral liquid described in the above technical solution in the quality control of Ganoderma lucidum oral liquid.

[0062] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0063] In the following examples, ganoderic acid A reference standard was purchased from the National Institutes for Food and Drug Control, batch number 111985-201601, specification 20mg / vial.

[0064] Preparation of Ganoderma lucidum oral liquid extract (numbered S4): Take 300 parts of Ganoderma lucidum, crush it into coarse powder, soak it in water for 12 hours, decoct it three times. The first time, add 8 times the amount of water and decoct for 5 hours. The second time, add 6 times the amount of water and decoct for 2 hours. The third time, add 4 times the amount of water and decoct for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to a clear paste with a relative density of 1.06~1.08 (80℃) (i.e., Ganoderma lucidum polysaccharide extract).

[0065] Preparation of Ganoderma lucidum oral liquid: 300 parts of Ganoderma lucidum polysaccharide extract, 200 parts of sucrose, 15 parts of stevioside, and 2 parts of sodium benzoate were dissolved in water and stirred well. Water was added to a final volume of 1000 parts, and the mixture was stirred well to obtain the Ganoderma lucidum oral liquid. The batch numbers of the prepared Ganoderma lucidum oral liquid are: 302240101 (numbered S1), 302240102 (numbered S2), and 302240103 (numbered S3).

[0066] Preparation of ganoderic acid A reference solution: Accurately weigh 1.92 mg of ganoderic acid A reference standard, place it in a 5 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well to obtain ganoderic acid A reference solution.

[0067] Preparation of the terpene test solution (for the detection of ganoderic acid A): Accurately measure 5 mL of Ganoderma lucidum oral liquid and place it in a 10 mL volumetric flask. Dilute with water to the mark, shake well, and centrifuge at 10000 r / min for 10 min. The supernatant solution is the terpene test solution. The batch number of the Ganoderma lucidum oral liquid used for the terpene test solutions in Comparative Examples 1-6 and Example 1 is 302240101.

[0068] Ganoderma lucidum polysaccharide standard: dextran molecular weight standard kit, purchased from the National Institutes for Food and Drug Control, batch number 140637-646-201203, specification 100mg / vial.

[0069] Preparation of Ganoderma lucidum polysaccharide test solution: 0.5g of Ganoderma lucidum oral liquid (batch number 302240102) was placed in an Erlenmeyer flask and dissolved in 10mL of hot water at 95~100℃. Then, 150mL of ethanol was added and mixed thoroughly. The mixture was allowed to stand at 4℃ for 12h, centrifuged at 4000r / min for 10min, and the supernatant was discarded. The lower layer of crude polysaccharide was washed twice with ethanol and the ethanol was removed at 80℃. 25mL of mobile phase was added to the remaining residue to dissolve it. The mixture was transferred to a volumetric flask, shaken well, and filtered through a 0.45μm filter membrane. The filtrate was the Ganoderma lucidum polysaccharide test solution.

[0070] The sulfuric acid is 98% sulfuric acid by mass.

[0071] Anthrone sulfate solution: Accurately weigh 0.1 g of anthrone, place it in a 100 mL volumetric flask, add 98 wt% sulfuric acid to dissolve and dilute to the mark, shake well to obtain anthrone sulfate solution.

[0072] Comparative Example 1 The HPLC chromatographic conditions described in Section 6.1 of the literature (Guo Zhongyuan, Hao Danli, Xie Ran, et al. Preparation and quality analysis methods of standard decoction of Ganoderma lucidum slices [J]. Chinese Journal of Traditional Chinese Medicine, 2021, 36(10):6178-6183.) were adopted, as follows: chromatographic column: Thermo-C 18Chromatographic column (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile (A) - 0.1% acetic acid (B); gradient elution (0~20 min, 72%~68% B; 20~40 min, 68%~64% B); flow rate: 1.0 mL / min; column temperature: 30 ℃; injection volume: 10 μL; detection wavelength: 256 nm; theoretical plate number calculated based on ganoderic acid A should not be less than 3000.

[0073] Table 1 Gradient elution program

[0074] The ganoderic acid A reference solution and the terpenoid test solution were analyzed by HPLC. Figure 1 This is the HPLC chromatogram of ganoderic acid A reference solution. Figure 2 The image shows the HPLC chromatogram of the terpene test solution. By comparing the two HPLC chromatograms, it can be seen that ganoderic acid A eluted at 3.087 min, which is too fast, and the chromatographic peaks of the sample could not be effectively separated.

[0075] Comparative Example 2 The only difference from Comparative Example 1 is that the chromatographic column is a Welch C10. 18 (4.6mm × 250mm, 5μm). Test results: The HPLC chromatogram of the terpene ganoderic acid A reference solution is shown below. Figure 3 The HPLC chromatogram of the ganoderic acid A reference solution showed that ganoderic acid A did not produce a peak.

[0076] Comparative Example 3 The HPLC chromatographic conditions in Section 2.4.1 of the literature ([1] Li Zhenyu, Pan Xiaojun, Liu Xiaoxia, et al. Study on quality standard of Ganoderma lucidum decoction [J]. Journal of Chinese Traditional Medicine Information, 2022, 29(2):98-103.) were adopted as follows: Agilent SB C18 column (2.1mm×150mm, 1.8μm) was used, the mobile phase was acetonitrile (A)-0.075% phosphoric acid solution (B), gradient elution (0~34min, 25%~26.5%A; 34~58min, 26.5%~38.5%A; 58~64min, 38.5%A), column temperature 25℃, flow rate 0.3mL / min, detection wavelength 257nm, and injection volume 1μL.

[0077] The ganoderic acid A reference solution and the terpenoid test solution were analyzed by HPLC.

[0078] Test results: Figure 4 This is the HPLC chromatogram of the terpene ganoderic acid A reference solution. As can be seen from the HPLC chromatogram of the ganoderic acid A reference solution, ganoderic acid A does not produce a peak.

[0079] Comparative Example 4 The only difference from Comparative Example 3 is that the chromatographic column is an Agilent SB C. 18 (4.6mm×250mm, 5μm), gradient elution program is shown in Table 2.

[0080] Table 2 Gradient elution program

[0081] Test results: Figure 5 The image shows the HPLC chromatogram of the terpene ganoderic acid A reference solution. By comparing the HPLC chromatogram of the ganoderic acid A reference solution, it can be seen that ganoderic acid A does not produce a peak.

[0082] Comparative Example 5 Referring to the chromatographic conditions of the reference (Sun Guoqiang, Dong Jinying, Cao Fahao. Analysis of active components of Ganoderma lucidum mycelium water extract [J]. Edible Fungi, 2023, 45(6):13-16.), the chromatographic column was JADE-PAK® ODS-AQ (5 μm, 250×4.6 mm), the mobile phase was acetonitrile (A)-0.1% formic acid aqueous solution (B), the mobile phase flow rate was 1.0 mL / min, the column temperature was 30℃, the injection volume was 10 μL, the detection wavelength was 256 nm, and the gradient elution program is shown in Table 3.

[0083] Table 3 Gradient elution program

[0084] The ganoderic acid A reference solution and the terpenoid test solution were analyzed by HPLC.

[0085] Test results: By comparing the HPLC chromatograms of the terpene test solution and the ganoderic acid A reference solution, it can be seen that the test solution has a chromatographic peak at the same position as the ganoderic acid A reference standard.

[0086] Example 1 HPLC chromatographic conditions: Agilent high-performance liquid chromatograph, Zafex C10 column. 18 (4.6 mm × 250 mm, 5 μm); mobile phase A is acetonitrile, mobile phase B is 0.1 vol% formic acid aqueous solution; mobile phase flow rate is 1.0 mL / min; column temperature is 30 ℃; injection volume is 10 μL; detection wavelength is 256 nm; gradient elution program is shown in Table 4.

[0087] Table 4 Gradient elution program

[0088] The ganoderic acid A reference solution and the terpenoid test solution were analyzed by HPLC. Figure 6 This is the HPLC chromatogram of ganoderic acid A reference solution. Figure 7The HPLC chromatogram of the terpene test solution shows that ganoderic acid A reference standard eluted at 41.173 min, while ganoderic acid A in the terpene test solution eluted at 41.207 min. The peaks in the terpene test solution were well separated. Therefore, the HPLC detection method provided in this embodiment can be used as a method for determining the fingerprint spectrum of Ganoderma lucidum oral liquid.

[0089] Example 2 Sample fingerprint analysis.

[0090] Ganoderma lucidum acid oral solution batch numbers: 302240101 (S1), 302240102 (S2), 302240103 (S3).

[0091] Terpenoid test solutions were prepared from three batches of Ganoderma lucidum oral liquid.

[0092] Three batches of terpenoid test solutions were subjected to HPLC testing according to the HPLC conditions of Example 1.

[0093] Figure 8 The superimposed HPLC chromatograms of the test solutions prepared from three batches of Ganoderma lucidum oral liquid show that the HPLC chromatograms of the three batches of Ganoderma lucidum oral liquid samples are... Figure 1 This indicates that the preparation process of Ganoderma lucidum oral liquid in this invention is stable.

[0094] Fingerprint chromatograms of three batches of Ganoderma lucidum acid oral solution (302240101, 302240102, and 302240103) were compared. The chromatograms were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)" to generate control chromatograms. The results are shown below. Figure 9 (Comparison of superimposed HPLC chromatograms of three batches of Ganoderma lucidum oral liquid and the chromatogram of Ganoderma lucidum oral liquid extract determination) Figure 10 (Comparison chromatograms generated from 3 batches of Ganoderma lucidum oral liquid), Table 5 and Table 6 show that the similarity of the HPLC comparison chromatograms of the three batches of Ganoderma lucidum oral liquid is greater than 0.9, and the 3 batches of Ganoderma lucidum acid oral liquid can generate comparison chromatograms. Figures 9-10 The “(20)” indicates that a total of 20 characteristic component peaks were collected and compared.

[0095] Table 5. Comparison of fingerprint similarity data among three batches of Ganoderma lucidum oral liquid.

[0096] Table 6. Comparison of fingerprint similarity data among three batches of Ganoderma lucidum oral liquid.

[0097] Example 3 Ganoderma lucidum polysaccharide molecular weight distribution Gel chromatography conditions: The chromatographic column consisted of Monomix MC10-SEC (7.8 mm × 300 mm, 10 μm, 1000 Å) and Monomix MC10-SEC (7.8 mm × 300 mm, 10 μm, 300 Å) in series; the detection cell temperature was 40 °C; the column temperature was 40 °C; the injection volume was 50 μL; the mobile phase was 0.2 mol / L sodium chloride aqueous solution; and the mobile phase flow rate was 0.5 ml / min.

[0098] (1) Drawing the standard curve Standards: The molecular weights of the dextran standards in the dextran molecular weight standard kit are shown in Table 7. In Tables 7 and 8, D0 to D8 and D2000 represent the numbers of the dextran standards with different molecular weights.

[0099] Table 7 Preparation of single dextran standard solutions for molecular weight distribution determination

[0100] Preparation of dextran mixed standard solution: Take 0.5 mL of each of the 10 single dextran standard solutions in Table 7 and mix them to obtain the dextran mixed standard solution.

[0101] The dextran mixed standard solution was analyzed by gel chromatography. The elution times of each dextran standard in the dextran mixed standard solution are shown in Table 8.

[0102] Table 8 Peak times of each dextran standard

[0103] Plot a standard curve with retention time on the x-axis and molecular weight on the y-axis.

[0104] The test solution of Ganoderma lucidum polysaccharide and the oral extract of Ganoderma lucidum were detected by gel chromatography.

[0105] Figure 11 The gel chromatogram of the test solution of Ganoderma lucidum polysaccharides shows that, based on the retention time, most of the Ganoderma lucidum polysaccharides in the oral liquid have a molecular weight of less than 180.

[0106] The gel chromatograms of Ganoderma lucidum oral liquid extract and three batches of Ganoderma lucidum polysaccharide test solutions were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)". The results are shown in the figure. Figure 12 (Comparison of gel chromatograms of Ganoderma lucidum oral liquid extract and three batches of Ganoderma lucidum polysaccharide test solutions) Figure 13 (Comparison chromatograms of Ganoderma lucidum oral liquid extract and 3 batches of Ganoderma lucidum polysaccharide test solutions), Table 9 and Table 10. It can be seen that the gel chromatograms of the 3 batches of Ganoderma lucidum oral liquid and Ganoderma lucidum oral liquid extract are similar. Figures 12-13 The “(5)” indicates that a total of 5 characteristic component peaks were collected and compared.

[0107] Table 9. Comparison of molecular weight distribution of polysaccharides from three batches of Ganoderma lucidum oral liquid samples and Ganoderma lucidum oral liquid extract.

[0108] Table 10. Comparison of molecular weight distribution similarity between three batches of Ganoderma lucidum oral liquid samples and Ganoderma lucidum oral liquid extract polysaccharides.

[0109] Example 4 The content of Ganoderma lucidum polysaccharides in Ganoderma lucidum oral liquid was determined by the sulfuric acid anthrone method.

[0110] Preparing a series of glucose reference solutions: Accurately weigh anhydrous glucose reference standard, dissolve and dilute with water to obtain a glucose reference standard stock solution with a concentration of 0.12 mg / mL per mL. Accurately measure 0.0, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, and 1.2 mL of the glucose reference standard stock solution, accurately add water to 2.0 mL respectively, quickly and accurately add 6 mL of anthrone sulfate solution, immediately shake well and time for 15 min, then immediately cool in an ice bath for 15 min to obtain a series of glucose reference solutions.

[0111] Blank solution: Accurately measure 2.0 mL of water, quickly and accurately add 6 mL of anthrone sulfate solution, shake well immediately and set a timer for 15 min, then immediately cool in an ice bath for 15 min.

[0112] Using spectrophotometry, the absorbance of a series of glucose standard solutions and blank solutions at a wavelength of 625 nm was measured, and A was obtained. 样品 and A 空白 , with ΔA (ΔA=A 样品 -A 空白 Plot a standard curve with y = 0.5 on the ordinate and glucose concentration on the x = 0.5.

[0113] Test solution: Accurately measure 0.2 mL of Ganoderma lucidum oral liquid, place it in a centrifuge tube, add 50 mL of anhydrous ethanol, vortex for 1 min, refrigerate and let stand for 0.5 h, centrifuge at 3500 r / min for 5 min, and discard the ethanol liquid; add 50 mL of anhydrous ethanol to the residue, vortex for 1 min, refrigerate and let stand for 0.5 h, centrifuge at 3500 r / min for 5 min, and discard the ethanol liquid; add 50 mL of anhydrous ethanol to the residue, vortex for 1 min, refrigerate and let stand for 0.5 h, centrifuge at 3500 r / min for 5 min, and discard the ethanol liquid; evaporate the residue in an 80℃ water bath until there is no alcohol odor, dissolve and dilute with water to 25 mL, shake well, filter, and obtain the test solution.

[0114] Test solution: Accurately measure 2 mL of the test solution, quickly and accurately add 6 mL of anthrone sulfate solution, shake well immediately and let stand for 15 min, then immediately cool in an ice bath for 15 min to obtain the test solution.

[0115] The absorbance of the test solution and the blank solution was measured at a wavelength of 625 nm using spectrophotometry, and A was obtained respectively. 样品 and A 空白 , ΔA (ΔA=A 样品 -A 空白 Substitute the values ​​into the standard curve to obtain the content of anhydrous glucose in the test solution, and calculate the content of Ganoderma lucidum polysaccharides in the Ganoderma lucidum oral liquid.

[0116] Limit: Anhydrous glucose (C6H) in Ganoderma lucidum oral liquid 12 The content of O6 shall not be less than 3.0 mg / mL.

[0117] Example 5 Standard curve Weigh 12.48 mg of anhydrous glucose reference standard and place it in a 100 mL volumetric flask. Dissolve and dilute to the mark with water, shake well, and accurately measure 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL into stoppered test tubes respectively. Accurately add water to 2.0 mL for each tube, and immediately add 6 mL of anthrone sulfate. Shake well immediately and let stand for 15 min. Then immediately cool in an ice bath for 15 min. Using the corresponding reagents as blanks, perform spectrophotometric experiments, measuring the absorbance at a wavelength of 625 nm. Plot a standard curve with absorbance as the ordinate and glucose concentration as the abscissa (see...). Figure 14 ).

[0118] Table 11 Standard Curve Determination Results—Anthrone Sulfate Method

[0119] Comparative Example 6 Blank solution: Take 1.0 mL of water, accurately add 2.0 mL of 4% phenol aqueous solution (w / v) (abbreviated as 4% phenol), shake well, accurately add 7.0 mL of sulfuric acid, keep warm at 60°C for 30 min, take it out, cool it immediately with running water, let it stand for 10 min, and measure the absorbance at 490 nm according to Example 4.

[0120] Construction of the standard curve: Take 10.07 mg of anhydrous glucose reference standard dried to constant weight at 105℃, place it in a 10 mL volumetric flask, add water to the mark, and shake well. Accurately measure 2.5 mL, place it in a 20 mL volumetric flask, add water to the mark, and shake well. Accurately measure 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL, and place them in stoppered test tubes respectively. Add water to 1.0 mL for each tube, accurately add 2.0 mL of 4% phenol for each tube, shake well, and accurately add 7.0 mL of sulfuric acid. Incubate at 60℃ for 30 min, remove, immediately cool with running water, and let stand for 10 min to obtain the test solution for the standard curve. Test the absorbance of each solution at 490 nm according to Example 4 and construct the standard curve (see Example 4). Figure 15 ).

[0121] Preparation of the test solution: Accurately measure 0.2 mL of Ganoderma lucidum oral liquid and place it in a 50 mL centrifuge tube. Quickly add 50 mL of ethanol and shake vigorously. Let stand for 1 h, centrifuge at 3500 r / min for 15 min, discard the supernatant, evaporate the solvent from the residue at 80℃, and accurately add 10.0 mL of water to dissolve it to obtain the test solution.

[0122] Sample testing: Accurately measure 1.0 mL of the test solution and place it in a stoppered test tube. Accurately add 2.0 mL of 4% phenol, shake well, and accurately add 7.0 mL of sulfuric acid. Incubate at 60°C for 30 min, remove, and immediately cool with running water. Let stand for 10 min to obtain the test solution. Test the absorbance at 490 nm according to Example 4 and calculate the Ganoderma lucidum polysaccharide content.

[0123] Table 12 Preparation and Measurement Results of Standard Curve

[0124] The experimental results are shown in Table 13. It can be seen that the absorbance data of the same sample varied greatly, and the repeatability was extremely poor.

[0125] Table 13 Results of repeatability tests (n=9)

[0126] Comparative Example 7 Method screening 1—Sulfuric acid phenol method for determination Standard curve: Accurately weigh 10 mg of glucose reference standard, place it in a 100 mL volumetric flask, dissolve and dilute with water to the mark, shake well, accurately measure 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL, add water to 1.0 mL respectively, add 2 mL of 4% phenol and 7 mL of sulfuric acid respectively, shake well to obtain the test solution for the standard curve.

[0127] Test solution: Measure 0.2 mL of Ganoderma lucidum oral liquid, place it in a centrifuge tube, add 50 mL of anhydrous ethanol, vortex for 1 min, refrigerate and let stand for 0.5 h, centrifuge at 3500 r / min for 5 min, and discard the ethanol liquid; add 50 mL of anhydrous ethanol to the obtained residue, vortex for 1 min, refrigerate and let stand for 0.5 h, centrifuge at 3500 r / min for 5 min, and discard the ethanol liquid; add 50 mL of anhydrous ethanol to the obtained residue, vortex for 1 min, refrigerate and let stand for 0.5 h, centrifuge at 3500 r / min for 5 min, and discard the ethanol liquid; evaporate the residue to dryness in an 80℃ water bath, dissolve and dilute with water to 25 mL, shake well, filter, and obtain the test solution.

[0128] Accurately measure 1 mL of the above test solution, add 2 mL of 4% phenol and 7 mL of sulfuric acid, shake well to obtain the test solution.

[0129] Repeatability test: Six test solutions were prepared, and the absorbance of each solution at a wavelength of 490 nm was tested according to Example 4. A standard curve was plotted, the content of Ganoderma lucidum polysaccharides in Ganoderma lucidum oral liquid was calculated, and a repeatability test was conducted.

[0130] The experimental results are shown in Table 14. It can be seen that the average value is 5.3 mg / mL and the RSD% is 9.3% (>8%). The repeatability of the detection method provided in this comparative example is generally poor.

[0131] Table 14 Repeatability Test Results—Sulfuric Acid Phenol Method

[0132] Example 6 Screening of detection methods: 2-anthrone sulfate method for determination Standard curve: Accurately weigh 12 mg of glucose reference standard, place it in a 100 mL volumetric flask, dissolve and dilute with water to the mark, shake well, accurately measure 0, 0.4, 0.6, 0.8, 1.0, and 1.2 mL, add water to 2.0 mL respectively, and quickly and accurately add 6 mL of anthrone sulfate solution to each, shake well immediately and let stand for 15 min, then immediately cool in an ice bath for 15 min to obtain the standard curve test solution.

[0133] Test solution: Measure 0.2 mL of Ganoderma lucidum oral liquid, place it in a centrifuge tube, add 50 mL of anhydrous ethanol, vortex for 1 min, refrigerate and let stand for 0.5 h, centrifuge at 3500 r / min for 5 min, and discard the ethanol liquid; add 50 mL of anhydrous ethanol to the residue, vortex for 1 min, refrigerate and let stand for 0.5 h, centrifuge at 3500 r / min for 5 min, and discard the ethanol liquid; add 50 mL of anhydrous ethanol to the residue, vortex for 1 min, refrigerate and let stand for 0.5 h, centrifuge at 3500 r / min for 5 min, and discard the ethanol liquid; evaporate the residue to dryness in an 80℃ water bath, dissolve and dilute with water to 25 mL, shake well, filter, and obtain the test solution.

[0134] Accurately measure 2 mL of the test solution, add 6 mL of anthrone sulfuric acid solution, shake well, let stand for 15 min, and then immediately cool in an ice bath for 15 min to obtain the test solution.

[0135] Repeatability test: Six solutions were prepared according to the method for preparing the test solution. The absorbance of each solution at a wavelength of 625 nm was tested according to Example 4, and a standard curve was plotted. The content of Ganoderma lucidum polysaccharides in the Ganoderma lucidum oral liquid was calculated. The results are shown in Table 15. It can be seen that the average value of the six content data points for repeatability is 5.4 mg / mL, and the RSD% is 5.3% < 8%. This indicates that the detection method provided in this example has good repeatability.

[0136] Table 15 Repeatability Test Results—Anthrone Sulfate Method

[0137] Intermediate precision test: The above repeatability test was conducted by different personnel. The results are shown in Table 16. It can be seen that the average value of the six content data points for intermediate precision was 4.7 mg / mL, and the RSD% was 7.3% < 8%. The intermediate precision is good.

[0138] Table 16 Intermediate Precision Test Results—Anthrone Sulfate Method

[0139] Summary of repeatability and intermediate precision test results The repeatability and intermediate precision data of 12 samples were summarized and the results are shown in Table 17. It can be seen that the RSD of the 12 samples was 9.4%.

[0140] Table 17 Precision Test Results—Anthrone Sulfate Method

[0141] Example 7 Sensitivity test—detection limit Preparing a series of glucose reference solutions: Accurately weigh 11.85 mg of glucose reference standard, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with water, and shake well; accurately measure 1.0 mL, place it in a 20 mL volumetric flask, and dilute to the mark with water to prepare standard curve test solution 1; continue to accurately measure 1.0, 1.5, 2.0, 2.5, and 30 mL, place them in 10 mL volumetric flasks respectively, and dilute to the mark with water to prepare standard curve test solutions.

[0142] Determination: Take 2.0 mL of the above test solution precisely, add 6 mL of anthrone sulfate solution precisely, shake well immediately and let stand for 15 min, then cool in an ice bath for 15 min immediately, and measure the absorbance at a wavelength of 625 nm immediately. Calculate the linear equation with respect to concentration and absorbance. The results are shown in Table 18.

[0143] Table 18 Detection Limit Determination Standard Curve Results—Anthrone Sulfate Method

[0144] The sample solution to be tested and the standard curve were prepared according to the method in Example 5 (see Example 5). Figure 16 The linear equation is: Y = 61.364X - 0.0089, with a correlation coefficient R = 0.996.

[0145] Detection limit: Calculated using the formula: 3.3SD / slope, with SD of 0.000707, the detection limit is 0.038 μg / mL, which accounts for 0.2% of the glucose content limit concentration of Ganoderma lucidum oral liquid (0.024 mg / mL). The method is relatively sensitive.

[0146] Limit of quantitation: Calculated using the formula 10SD / slope, with SD of 0.000707, the limit of detection was 0.115 μg / mL, which is 0.5% of the glucose content limit concentration of Ganoderma lucidum oral liquid (0.024 mg / mL). The method is relatively sensitive.

[0147] Example 8 Phenol-sulfuric acid process Blank solution: Accurately measure 5.0 mL of water and place it in a 50 mL volumetric flask. Add 10 mL of 4% phenol and 66 g of sulfuric acid. Shake well and incubate in a 60 °C water bath for 30 min. Remove the flask and immediately place it in an ice bath for 10 min to obtain the blank solution.

[0148] Construction of the standard curve: Anhydrous glucose standard dried to constant weight at 105℃ was dissolved in water to obtain a glucose standard stock solution with a concentration of 0.1 mg / mL. Accurately measure 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL of the glucose standard stock solution and place them in separate 50 mL volumetric flasks. Accurately add water to each flask to a final volume of 5.0 mL, add 10 mL of 4% phenol, and accurately add 66 g of sulfuric acid. Shake well and incubate in a 60℃ aqueous solution for 30 min. Remove from the solution and immediately place it in an ice bath for 10 min to obtain the test solution. Test the absorbance of each solution at 490 nm according to Example 5 and construct a standard curve (see Example 5). Figure 17 ).

[0149] Table 19 Standard Curve Determination Results—Sulfuric Acid-Phenol Method

[0150] Test solution: Accurately measure 0.2 mL of Ganoderma lucidum oral liquid and place it in a 50 mL centrifuge tube. Quickly add 30 mL of ethanol and shake at 200 r / min for 1 min (vortex mixing). Let it stand at low temperature (5℃) for 0.5 h, centrifuge at 3500 r / min for 5 min, discard the supernatant, add ethanol and repeat the above precipitation operation twice. Evaporate the solvent from the residue at 80℃, add water until dissolved, transfer all to a 25 mL volumetric flask, sonicate for 20 min, dilute to the mark with water, shake well, and obtain the test solution.

[0151] Accurately measure 5.0 mL of the test solution and place it in a 50 mL volumetric flask. Add 10 mL of 4% phenol and 66 g of sulfuric acid. Shake well and incubate in a 60°C water bath for 30 min. Remove the flask and immediately place it in an ice bath for 10 min to obtain the test solution. Test the absorbance of each solution at 490 nm according to Example 5 and calculate the content of Ganoderma lucidum polysaccharides in the Ganoderma lucidum oral liquid. The results are shown in Table 20.

[0152] Table 20 Results of Glucose Content Determination in Different Batch Samples - Phenol-Sulfuric Acid Method

[0153] In the detection methods provided by this invention, the anthrone sulfate method is slightly superior to the phenol sulfate method.

[0154] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of Ganoderma lucidum oral liquid, characterized in that, The fingerprint spectrum includes terpenoid fingerprint spectrum and Ganoderma lucidum polysaccharide fingerprint spectrum; The method for constructing the terpene fingerprint includes the following steps: The Ganoderma lucidum oral liquid was diluted with water to obtain a terpenoid test solution. The terpene test solution and the ganoderic acid A reference solution were respectively subjected to high performance liquid chromatography to obtain the liquid chromatograms of the test sample and the reference sample, respectively. The conditions for high-performance liquid chromatography (HPLC) detection include: a C18 column; a mobile phase system comprising mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is a 0.8-0.12% (v / v) formic acid aqueous solution; and a gradient elution method, wherein the gradient elution procedure is as follows: From 0 to 25 minutes, the volume fraction of the mobile phase A increased from 27-28.5% to 29-32%. Over 25-50 minutes, the volume fraction of mobile phase A increased from 29-32% to 37-41%. Over 50-60 minutes, the volume fraction of mobile phase A increased from 37-41% to 58-62%. Over 60-70 minutes, the volume fraction of mobile phase A decreased from 58-62% to 27-28.5%. The retention times of the liquid chromatography chromatogram of the test sample and the liquid chromatography chromatogram of the reference sample were compared to obtain the terpene fingerprint chromatogram. The method for constructing the Ganoderma lucidum polysaccharide fingerprint spectrum includes the following steps: Ganoderma lucidum oral liquid was dissolved in hot water and precipitated with ethanol to obtain crude Ganoderma lucidum polysaccharide; the crude Ganoderma lucidum polysaccharide was dissolved in sodium chloride aqueous solution with a concentration of 0.1~0.3mol / L to obtain Ganoderma lucidum polysaccharide test solution; The fingerprint spectrum and the Ganoderma lucidum polysaccharide reference solution were subjected to gel chromatography to obtain gel chromatograms of the test sample and the reference sample, respectively; the mobile phase for gel chromatography included an aqueous sodium chloride solution with a concentration of 0.1~0.3 mol / L. The molecular weight of the test sample gel chromatography was compared with that of the reference sample gel chromatography to obtain the Ganoderma lucidum polysaccharide fingerprint.

2. The construction method according to claim 1, characterized in that, The conditions for high-performance liquid chromatography detection also include: the chromatographic column is a Zafex C10 ... 18 The column temperature is 25~35℃; the mobile phase flow rate is 0.8~1.2mL / min; the injection volume is 5~15μL; and the detection wavelength is 256nm.

3. The construction method according to claim 1, characterized in that, The volume ratio of the Ganoderma lucidum oral liquid to the diluting water is 1:0.5~1.5; The dilution process also includes centrifuging the diluted solution.

4. The construction method according to claim 1, characterized in that, The concentration of ganoderic acid A reference standard in the ganoderic acid A reference solution is 0.2~0.5 mg / mL, and the solvent includes methanol.

5. The construction method according to claim 1, characterized in that, The conditions for gel chromatography detection also include: the chromatographic column is a Monomix MC10-SEC column; the detection cell temperature is 35~45℃; the column temperature is 35~45℃; the mobile phase flow rate is 0.3~0.7mL / min; and the injection volume is 20~80μL.

6. The construction method according to claim 1, characterized in that, The ratio of the amount of Ganoderma lucidum oral liquid to hot water is 1g: 10~30mL; The ratio of the amount of Ganoderma lucidum oral liquid to the amount of ethanol used for precipitation is 1g: 200~400mL; The precipitation was carried out at a temperature of 2-6°C for 8-16 hours. The ratio of the amount of Ganoderma lucidum oral liquid to the sodium chloride aqueous solution for dissolving is 1g: 30~70mL; The precipitation process further includes: sequentially centrifuging the precipitated system, and washing the resulting solid components with ethanol to remove the ethanol.

7. The construction method according to claim 1, characterized in that, The Ganoderma lucidum polysaccharide reference solution contains a set of dextran molecular weight standards, which includes dextran with molecular weights of 180, 2700, 5250, 9750, 13050 and 36800. The solvent in the Ganoderma lucidum polysaccharide reference solution includes an aqueous sodium chloride solution with a concentration of 0.1~0.3 mol / L.

8. A method for detecting the content of Ganoderma lucidum polysaccharides in Ganoderma lucidum oral liquid, characterized in that, Includes the following steps: Ganoderma lucidum oral liquid and ethanol are mixed and precipitated to obtain a precipitate. The precipitate is then dissolved in water to obtain a test solution. The precipitation is carried out 3 to 5 times. The absorbance of the test solution was tested, and the test results of the Ganoderma lucidum polysaccharide content in the Ganoderma lucidum oral liquid were obtained according to the absorbance and the absorbance-concentration standard curve; the absorbance-concentration standard curve used the glucose concentration as the independent variable and the absorbance as the dependent variable.

9. The detection method according to claim 8, characterized in that, The precipitation temperature is 2~8℃, and the precipitation time for a single precipitation is 30~60min; The dissolution is carried out under ultrasonic conditions, and the dissolution time is 15-30 minutes. The absorbance testing methods include the phenol-sulfuric acid method or the sulfuric acid-anthrone method.

10. The application of the method for constructing the fingerprint spectrum of Ganoderma lucidum oral liquid according to any one of claims 1 to 7 or the method for detecting the content of Ganoderma lucidum polysaccharides in Ganoderma lucidum oral liquid according to claim 8 or 9 in the quality control of Ganoderma lucidum oral liquid.

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