HPLC (High Performance Liquid Chromatography) fingerprint spectrum research and multi-component content analysis method of ganoderma lucidum green tea

Abstract

The invention relates to the technical field of lucid ganoderma and green tea capsule analysis, and discloses a lucid ganoderma and green tea capsule HPLC (High Performance Liquid Chromatography) fingerprint spectrum research and multi-component content analysis method. The components are epigallocatechin, catechin, ganoderic acid C2, ganoderic acid A, ganoderic acid D, ganoderic acid D, ganoderic acid F and ganoderic acid G respectively. According to the HPLC fingerprint spectrum research and multi-component content analysis method for the ganoderma lucidum and green tea capsules, a sample is subjected to sample introduction analysis by adopting a high performance liquid chromatography, a fingerprint spectrum is made by using traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system software, similarity evaluation is performed, and quality evaluation is performed on the ganoderma lucidum and green tea capsule sample by combining CA, PCA and OPLS-DA. A ganoderma lucidum green tea capsule HPLC fingerprint spectrum is established, a chemical mode recognition analysis method is combined for comprehensive analysis of data, and quantitative analysis of epigallocatechin, ganoderic acid C2 and ganoderic acid A in the ganoderma lucidum green tea capsule is completed.

Description

Technical Field

[0001] The invention relates to the technical field of ganoderma lucidum green tea capsule analysis, in particular to HPLC fingerprint research and multi-component content analysis method of ganoderma lucidum green tea capsule. Background Art

[0002] Lingzhi Green Tea Capsule is a health care medicine that can significantly improve the body's immunity, enhance the activity of immune cells such as superoxide dismutase, improve the phagocytic ability of macrophages, promote the proliferation of lymphocytes, increase the number of antibody-producing cells, and improve the body's cellular immunity and humoral immunity.

[0003] At present, when analyzing the component content of a drug, a method of establishing an HPLC fingerprint is usually adopted. However, there is currently a lack of a method for studying the HPLC fingerprint of a Ganoderma lucidum green tea capsule and accurately analyzing the content of multiple components. Therefore, the present invention provides an HPLC fingerprint study and a multi-component content analysis method for a Ganoderma lucidum green tea capsule, which can achieve qualitative and quantitative analysis of each component in the Ganoderma lucidum green tea capsule, and provide a strong basis for measuring product quality. Summary of the invention

[0004] (I) Technical solution

[0005] The present invention provides the following technical solution: HPLC fingerprint study and multi-component content analysis method of Ganoderma lucidum green tea capsule, comprising the following steps:

[0006] S1. Prepare solution

[0007] S101. Weigh epigallocatechin, catechin, ganoderic acid C2, ganoderic acid G, ganoderic acid A, ganoderic acid D, ganoderic acid D and ganoderic acid F respectively, add methanol to fully dissolve them respectively, make up to the scale, shake evenly, and obtain a reference solution with a specific mass concentration.

[0008] S102. Grind the Ganoderma lucidum green tea extract sample, weigh it accurately, put it in a conical flask, accurately add methanol solution, weigh it, ultrasonicate it for 30 minutes, let it cool, weigh it again, make up the lost amount with methanol solution, shake it well, filter it, and filter the filtrate with a 0.45μm microporous filter membrane to obtain the test solution.

[0009] S103, according to the prescription composition of Ganoderma lucidum green tea capsules, prepare single herb samples of Ganoderma lucidum and green tea respectively, and make each single herb sample solution according to the test sample preparation method in step S102.

[0010] S2. Establish HPLC fingerprint

[0011] S201, precision, repeatability and stability test

[0012] According to the test regulations, the test target solution was selected, and the appropriate injection procedure was set. Epigallocatechin was used as the reference peak, and the relevant data were screened to calculate the common peak RSD value and record it in the table.

[0013] S202, establishing fingerprint map and similarity evaluation

[0014] Take 10 batches of Ganoderma lucidum green tea test solution, inject and measure according to the chromatographic conditions, record the chromatogram, and make sure that the peak area <10 6 After the chromatographic peaks are screened out, they are imported into the similarity evaluation software of Chinese medicine chromatographic fingerprint. S9 is used as the reference spectrum, the median method is adopted, the time width is set to 0.1, multi-point correction, automatic peak matching, and a control fingerprint spectrum (R) is generated. According to the chromatographic conditions, each sample solution in step S1 is taken for sampling analysis, and a chromatogram is drawn. The common peaks are identified by calibrating the chromatographic peaks of the fingerprint spectra of 10 batches of samples.

[0015] S3. Chemical pattern recognition analysis

[0016] S301, Cluster Analysis CA

[0017] The common peak area identified in step S202 was used as a variable and imported into IBM SPSS Statistics 27 software. The intergroup linkage method was used and CA was performed using the squared Euclidean distance as the measure.

[0018] S302, principal component analysis PCA

[0019] The related variables were converted into linear unrelated variables by means of data dimensionality reduction to reduce the number of variables and improve the analysis efficiency. The common peak areas of the 10 batches of Ganoderma Lucidum Green Tea Capsule samples identified in step S202 were imported into IBM SPSS Statistics 27 software for PCA, and the results were output and recorded. The common peak area was then imported into SIMCA software as a variable to intuitively reflect the characteristic distribution between sample batches and record them.

[0020] S303, Orthogonal Partial Least Squares-Discriminant Analysis OPLS-DA

[0021] The common peak areas of the 10 batches of Ganoderma Lucidum Green Tea Capsule samples identified in step S202 were imported into SIMCA software for OPLS-DA.

[0022] S4. Determination of multi-component content

[0023] S401. Preparation of mixed reference solution

[0024] Accurately weigh appropriate amounts of epigallocatechin, ganoderic acid C2, and ganoderic acid A standard products, respectively, place them in the same 10 mL volumetric flask, add methanol to dissolve, make up to volume, and shake well to obtain a mixed reference solution of epigallocatechin, ganoderic acid C2, and ganoderic acid A.

[0025] S402, Linear Relationship Investigation

[0026] The reference solution prepared in step S401 is sampled and analyzed, and a linear regression study is performed with the reference concentration as the horizontal axis (x) and the peak area as the vertical axis (y), and the results are recorded in a table.

[0027] S403, precision, repeatability and stability inspection

[0028] According to the specific requirements of the test, the target solution required for the test was determined, and the injection procedure was set. When calculating the RSD value of the common peak, epigallocatechin was used as the reference peak, the corresponding data was selected, and the results were recorded in a table.

[0029] S404, sample recovery test

[0030] Accurately weigh 6 portions of Ganoderma lucidum green tea capsule samples, add epigallocatechin, ganoderic acid A, and ganoderic acid C2 in a ratio of 100% of the sample content, follow the method described in step S102 to prepare the test solution, and inject the sample for analysis to obtain the specific value of the sample recovery rate, and record it in the table.

[0031] S405, sample content determination

[0032] Ten batches of Ganoderma lucidum green tea capsule samples were prepared into test sample solutions according to the method in step S102, and the samples were injected and analyzed under chromatographic conditions. The contents of epigallocatechin, ganoderic acid C2, and ganoderic acid A in each batch of samples were calculated and recorded in a table.

[0033] S5. Results and Discussion

[0034] According to the method of steps S1 to S4, the obtained data are analyzed.

[0035] Preferably, in step S101, the mass concentrations of epigallocatechin, catechin, ganoderic acid C2, ganoderic acid G, ganoderic acid A, ganoderic acid D, ganoderic acid D and ganoderic acid F obtained are 0.51, 2.39, 0.10, 0.12, 0.34, 0.13, 0.25 and 0.13 mg / mL, respectively.

[0036] Preferably, the chromatographic conditions in steps S202 and S405 are as follows: chromatographic column: WondaSil C18-WR (250 mm×4.6 mm, 5 μm). Mobile phase: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), low pressure gradient elution, flow rate: 1.0 mL·min-1. Column temperature: 30° C. Detection wavelength: 254 nm. Injection volume: 10 μL.

[0037] Preferably, in step S301, when the square Euclidean distance is 5, 10 batches of samples can be divided into 2 categories, samples 1 and 2 obtained after extraction of medicinal materials from the same supplier are grouped into the first category, and samples 3-10 obtained after extraction of medicinal materials from another supplier are grouped into the second category.

[0038] (II) Beneficial effects

[0039] Compared with the prior art, the present invention provides a HPLC fingerprint study and multi-component content analysis method for Ganoderma lucidum green tea capsules, which has the following beneficial effects:

[0040] The HPLC fingerprint study and multi-component content analysis method of Ganoderma lucidum green tea capsule adopts high performance liquid chromatography to analyze the samples, uses traditional Chinese medicine chromatographic fingerprint similarity evaluation system software to prepare fingerprints and conduct similarity evaluation, combines CA, PCA, and OPLS-DA to evaluate the quality of Ganoderma lucidum green tea capsule samples, establishes the HPLC fingerprint of Ganoderma lucidum green tea capsule, combines the chemical pattern recognition analysis method to conduct comprehensive analysis of the data, completes the quantitative analysis of epigallocatechin, ganoderic acid C2, and ganoderic acid A in Ganoderma lucidum green tea capsule, and the establishment of the HPLC fingerprint of Ganoderma lucidum green tea capsule and the multi-component content analysis method are accurate and stable, which lays a foundation for the quality control and evaluation of Ganoderma lucidum green tea capsule. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of HPLC fingerprints of 10 batches of Ganoderma lucidum green tea samples of the present invention;

[0042] Figure 2 This is a schematic diagram of the comparison of 10 batches of Ganoderma lucidum green tea capsule samples of the present invention;

[0043] Figure 3 HPLC charts of single herb Ganoderma lucidum, single herb green tea, mixed reference substance and Ganoderma lucidum green tea capsule samples of the present invention;

[0044] Figure 4 CA images of 10 batches of Ganoderma lucidum green tea samples of the present invention;

[0045] Figure 5 It is the PCA scree plot of the present invention;

[0046] Figure 6PCA score graphs of 10 batches of drugs of the present invention;

[0047] Figure 7 This is the OPLS-DA score diagram of the present invention;

[0048] Figure 8 This is the OPLS-DA VIP diagram of the present invention. DETAILED DESCRIPTION

[0049] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] See also Figure 1-8 The present invention provides a technical solution: HPLC fingerprint study and multi-component content analysis method of Ganoderma lucidum green tea capsule, comprising the following steps:

[0051] S1. Prepare solution

[0052] S101. Weigh epigallocatechin, catechin, ganoderic acid C2, ganoderic acid G, ganoderic acid A, ganoderic acid D, ganoderic acid D and ganoderic acid F respectively, add methanol to fully dissolve them, make up to the scale, shake evenly, and the mass concentrations of epigallocatechin, catechin, ganoderic acid C2, ganoderic acid G, ganoderic acid A, ganoderic acid D, ganoderic acid D and ganoderic acid F are 0.51, 2.39, 0.10, 0.12, 0.34, 0.13, 0.25 and 0.13 mg / mL respectively.

[0053] S102. Grind the Ganoderma lucidum green tea extract sample, weigh it accurately, put it in a conical flask, accurately add methanol solution, weigh it, ultrasonicate it for 30 minutes, let it cool, weigh it again, make up the lost amount with methanol solution, shake it well, filter it, and filter the filtrate with a 0.45μm microporous filter membrane to obtain the test solution.

[0054] S103, according to the prescription composition of Ganoderma lucidum green tea capsules, prepare single herb samples of Ganoderma lucidum and green tea respectively, and make each single herb sample solution according to the test sample preparation method in step S102.

[0055] S2. Establish HPLC fingerprint

[0056] S201, precision, repeatability and stability test

[0057] According to the test regulations, select the test target solution and set the appropriate injection procedure. Take epigallocatechin as the reference peak, screen the relevant data to calculate the common peak RSD value, and record it as shown in the small table.

[0058] Precision, repeatability and stability test results table

[0059]

[0060] S202, establishing fingerprint map and similarity evaluation

[0061] Take 10 batches of Ganoderma lucidum green tea test solution, inject and measure according to the chromatographic conditions, record the chromatogram, and make sure that the peak area <10 6 After the chromatographic peaks were screened out, they were imported into the Chinese medicine chromatographic fingerprint similarity evaluation software. S9 was used as the reference spectrum, the median method was used, the time width was set to 0.1, multi-point correction, automatic peak matching, and a reference fingerprint spectrum (R) was generated. The superimposed spectrum of the fingerprint spectrum of 10 batches of Ganoderma lucidum green tea samples and their reference fingerprint spectrum are shown in Figure 1 , 2 As shown. The similarity between the 10 batches of samples and R was calculated using the Chinese medicine chromatographic fingerprint similarity evaluation software to be 0.936-1, which is a high similarity, indicating that the quality of each batch of Lingzhi Green Tea Capsule samples is relatively consistent and stable. According to the chromatographic conditions, each sample solution in step S1 is taken for injection analysis, and a chromatogram is drawn, as shown in FIG. Figure 3 As shown, by calibrating the chromatographic peaks of the fingerprints of 10 batches of samples, 23 common peaks were identified. Among them, peaks 8-11 originated from green tea components, peaks 12-23 belonged to Ganoderma lucidum, and peaks 1-7 were determined to be miscellaneous peaks generated by the instrument. By comparing the chromatographic peaks and retention times of the mixed reference substances, 8 chromatographic peaks were identified, and 8 specific chromatographic peaks were successfully identified, namely epigallocatechin (peak 8), catechin (peak 9), ganoderic acid C2 (peak 12), ganoderic acid G (peak 13), ganoderic acid A (peak 17), ganoderic acid D (peak 19), ganoderic acid D (peak 20), and ganoderic acid F (peak 23).

[0062] S3. Chemical pattern recognition analysis

[0063] S301, Cluster Analysis CA

[0064] The common peak area identified in step S202 was used as a variable and imported into IBM SPSS Statistics 27 software. The intergroup linkage method was used and the squared Euclidean distance was used as the measure for CA. The results are as follows: Figure 4As shown in the figure. When the squared Euclidean distance is 5, 10 batches of samples can be divided into 2 categories. Samples 1 and 2 obtained after extraction from the same supplier's medicinal materials are clustered into the first category, and samples 3-10 obtained after extraction from another supplier's medicinal materials are clustered into the second category. This further shows that different suppliers of raw medicinal materials will lead to differences in samples between different batches.

[0065] S302, principal component analysis PCA

[0066] By means of data dimension reduction, the related variables were converted into linear unrelated variables to reduce the number of variables and improve the analysis efficiency. The 23 common peak areas of the 10 batches of Lingzhi Green Tea Capsule samples identified in step S202 were imported into IBM SPSSStatistics 27 software for PCA, and the results were output and recorded. As a result, 4 principal components with eigenvalues ​​> 1 were screened out, and the eigenvalues ​​were 15.096, 3.065, 2.124, and 1.107, respectively. The variance contribution rates were 65.637%, 13.327%, 9.233%, and 4.813%, respectively, and the cumulative variance contribution rate was 93.010%. As shown in the scree plot ( Figure 5 ), the slope is large when the four principal components appear, and it is relatively gentle when the subsequent components appear, which can explain that the four principal components effectively integrate the core information of all measurement indicators, thereby achieving the goal of dimensionality reduction. Then the common peak area is imported into the SIMCA software as a variable to intuitively reflect the characteristic distribution between sample batches and record it, such as Figure 6 As shown, the results are consistent with the CA results, samples 1 and 2 are divided into one category, and samples 3-10 are divided into one category.

[0067] S303, Orthogonal Partial Least Squares-Discriminant Analysis OPLS-DA

[0068] The common peak areas of the 10 batches of Lingzhi Green Tea Capsule samples identified in step S202 were imported into SIMCA software for OPLS-DA. Figure 7 As shown, the model R 2 X(cum)=0.864,R 2 Y(cum)=1,Q 2 =0.998, both greater than 0.5, indicating that the model fits more accurately and has good interpretability and predictability. Figure 8 As shown in the figure, under the condition that the VIP value is not less than 1, 12 differential standards were screened out with significant statistical significance. According to their importance, they are peak 9 (catechins), peak 10, peak 12 (ganoderic acid C2), peak 15, peak 13 (ganoderic acid G), peak 14, peak 11, peak 16, peak 19 (ganoderic acid D), peak 17 (ganoderic acid A), peak 18, and peak 22.

[0069] S4. Determination of multi-component content

[0070] S401. Preparation of mixed reference solution

[0071] Accurately weigh appropriate amounts of epigallocatechin, ganoderic acid C2, and ganoderic acid A standard products, respectively, place them in the same 10 mL volumetric flask, add methanol to dissolve, make up to volume, and shake well to obtain a mixed reference solution of epigallocatechin, ganoderic acid C2, and ganoderic acid A.

[0072] S402, Linear Relationship Investigation

[0073] The reference solution prepared in step S401 was sampled and analyzed, and a linear regression study was performed with the reference concentration as the abscissa (x) and the peak area as the ordinate (y), and the results were recorded in a table. The results are shown in the table below, and each component has a good linear relationship within the linear range.

[0074] Linear relationship investigation results table

[0075]

[0076] S403, precision, repeatability and stability inspection

[0077] According to the specific requirements of the test, the target solution required for the test was determined, and the injection procedure was set. When calculating the RSD value of the common peak, epigallocatechin was used as the reference peak, and the corresponding data was selected and the results were recorded in the table. The final results were in full compliance with the relevant regulations, and the specific data are shown in the following table.

[0078] Precision, repeatability and stability test results table

[0079]

[0080] S404, sample recovery test

[0081] Accurately weigh 6 portions of Ganoderma lucidum green tea capsule samples, add epigallocatechin, ganoderic acid A, and ganoderic acid C2 in a ratio of 100% of the sample content, follow the method described in step S102 to prepare the test solution, and inject the sample for analysis to obtain the specific value of the sample recovery rate, which is recorded as shown in the following table.

[0082]

[0083]

[0084] S405, sample content determination

[0085] Ten batches of Ganoderma lucidum green tea capsule samples were prepared into test solutions according to the method in step S102, and sampled and analyzed under chromatographic conditions. The contents of epigallocatechin, ganoderic acid C2, and ganoderic acid A in each batch of samples were calculated and recorded in the following table.

[0086] Sample content results table

[0087]

[0088] S5. Results and Discussion

[0089] According to the method of steps S1 to S4, the obtained data are analyzed.

[0090] The similarity evaluation software of Chinese medicine chromatographic fingerprint was used to calculate, and the overall similarity of the Lingzhi Green Tea Capsule samples was 0.936-1, showing a high similarity. This result shows that the Lingzhi Green Tea Capsule samples from different batches show high consistency and stability in quality. Therefore, it can be considered that the quality control results are reliable and have high credibility. The results of chemical pattern recognition analysis showed that the CA and PCA results were consistent, and the samples made from medicinal materials from different suppliers were divided into one category, which further verified the differences between samples. According to the variable importance projection diagram of OPLS-DA, 12 differential standards were screened out. The manufacturer can closely monitor the quality fluctuations of these 12 key components in the manufacturing process to ensure the stability and reliability of the product quality. The unknown components can be qualitatively and quantitatively analyzed using liquid chromatography-mass spectrometry.

[0091] The present invention identifies 8 components in total by comparing the ganoderma lucidum green tea test solution and the mixed reference solution, which are epigallocatechin, catechin, ganoderic acid C2, ganoderic acid A, ganoderic acid D, ganoderic acid D, ganoderic acid F, and ganoderic acid G. In order to show that the curative effect of ganoderma lucidum green tea capsule is the combination of ganoderma lucidum and green tea, the representative components of ganoderma lucidum and green tea are respectively selected for content determination. Among the 8 components, epigallocatechin and catechin belong to green tea and are the main components of green tea polyphenols. In the HPLC chromatogram, the separation degree of epigallocatechin is better than that of catechin, with higher resolution and reproducibility, and the content determination is more accurate, so epigallocatechin is selected for content determination. In addition to the above-mentioned components, the other six components are all derived from ganoderma lucidum, and they belong to ganoderma lucidum triterpenoid compounds. These compounds have multiple functions in vivo, such as anti-free radical, anti-inflammatory and antiviral effects, and they can also effectively kill cancer cells and have analgesic functions. These characteristics make the triterpenoids of Ganoderma lucidum have important application value in the field of traditional Chinese medicine. It can reduce cholesterol, prevent vascular sclerosis, and strengthen the liver's detoxification function. Therefore, the ganoderic acid C2 and ganoderic acid A with good chromatographic peak separation, high sensitivity and reproducibility were selected for content determination, and they were the difference indicators screened out from the OPLS-DA analysis of VIP results. The content determination results of these three components ranged from 18.05-19.11 mg / g, 2.41-2.46 mg / g, and 4.40-4.91 mg / g, respectively, and the RSD values ​​were 2.00%, 0.53%, and 0.18%, respectively, indicating that the content differences between samples were small, which can provide a basis for formulating content standards.

[0092] The chromatographic conditions in the present invention are as follows: chromatographic column: WondaSil C18-WR (250 mm×4.6 mm, 5 μm). Mobile phase: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), low pressure gradient elution, flow rate: 1.0 mL·min-1. Column temperature: 30°C. Detection wavelength: 254 nm. Injection volume: 10 μL. Elution conditions are shown in the following table.

[0093] Low pressure gradient elution conditions table

[0094]

[0095] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Study on HPLC fingerprint of Ganoderma lucidum green tea capsule and multi-component content analysis method, characterized in that: The following steps are involved: S1. Prepare solution S101, respectively weigh epigallocatechin, catechin, ganoderic acid C2, ganoderic acid G, ganoderic acid A, ganoderic acid D, ganoderic acid D and ganoderic acid F, respectively add methanol to fully dissolve them, dilute to the scale, shake evenly, and obtain a reference solution with a specific mass concentration; S102, grind the Ganoderma lucidum green tea extract sample, accurately weigh it, put it in a conical flask, accurately add the methanol solution, weigh it, ultrasonicate it for 30 minutes, let it cool, weigh it again, make up the lost amount with the methanol solution, shake it well, filter it, and filter the filtrate with a 0.45 μm microporous filter membrane to obtain the test solution; S103, preparing single herb samples of ganoderma lucidum and green tea respectively according to the prescription composition of ganoderma lucidum green tea capsules, and preparing single herb sample solutions according to the test sample preparation method in step S102; S2. Establish HPLC fingerprint S201, precision, repeatability and stability test According to the test regulations, select the test target solution and set the appropriate injection procedure. Take epigallocatechin as the reference peak, screen the relevant data to calculate the common peak RSD value, and record it in the table; S202, establishing fingerprint map and similarity evaluation Take 10 batches of Ganoderma lucidum green tea test solution, inject and measure according to the chromatographic conditions, record the chromatogram, and make sure that the peak area <10 6 After the chromatographic peaks are screened out, the chromatographic fingerprint similarity evaluation software of traditional Chinese medicine is introduced, and S9 is used as the reference spectrum. The median method is adopted, the time width is set to 0.1, multi-point correction, automatic peak matching, and a control fingerprint spectrum (R) is generated. According to the chromatographic conditions, each sample solution in step S1 is taken for sample analysis, and a chromatogram is drawn. The common peaks are identified by calibrating the chromatographic peaks of the fingerprint spectra of 10 batches of samples; S3. Chemical pattern recognition analysis S301, Cluster Analysis CA The common peak area identified in step S202 was used as a variable and imported into IBM SPSS Statistics 27 software, and CA was performed using the intergroup linkage method and the squared Euclidean distance as the measure; S302, principal component analysis PCA By means of data dimensionality reduction, the related variables are converted into linear unrelated variables to reduce the number of variables to improve the analysis efficiency. The common peak areas of the 10 batches of Lingzhi Green Tea Capsule samples identified in step S202 are imported into IBM SPSS Statistics27 software for PCA, and the results are output and recorded. The common peak areas are then imported into SIMCA software as variables to intuitively reflect the characteristic distribution between sample batches and record them; S303, Orthogonal Partial Least Squares-Discriminant Analysis OPLS-DA The common peak areas of the 10 batches of Ganoderma Lucidum Green Tea Capsule samples identified in step S202 were imported into SIMCA software for OPLS-DA; S4. Determination of multi-component content S401. Preparation of mixed reference solution Accurately weigh appropriate amounts of epigallocatechin, ganoderic acid C2, and ganoderic acid A standard substances, respectively, place them in the same 10 mL volumetric flask, add methanol to dissolve, make up to volume, and shake well to obtain a mixed reference solution of epigallocatechin, ganoderic acid C2, and ganoderic acid A; S402, Linear Relationship Investigation The reference substance solution prepared in step S401 is sampled and analyzed, and a linear regression study is performed with the reference substance concentration as the abscissa (x) and the peak area as the ordinate (y), and the obtained results are recorded in a table; S403, precision, repeatability and stability inspection According to the specific requirements of the test, the target solution required for the test was determined, and the injection procedure was set. When calculating the RSD value of the common peak, epigallocatechin was used as the reference peak, and the corresponding data was selected, and the results were recorded in the table; S404, sample recovery test Accurately weigh 6 portions of Lingzhi green tea capsule samples, add epigallocatechin, ganoderic acid A, and ganoderic acid C2 in a ratio of 100% of the sample content, follow the method described in step S102 to prepare the test solution, inject and analyze the sample, obtain the specific value of the sample recovery rate, and record it in a table; S405, sample content determination Prepare the test solution of 10 batches of Ganoderma lucidum green tea capsule samples according to the method in step S102, inject and analyze under chromatographic conditions, calculate the content of epigallocatechin, ganoderic acid C2, and ganoderic acid A in each batch of samples, and record them in a table; S5. Results and Discussion According to the method of steps S1 to S4, the obtained data are analyzed.

2. The HPLC fingerprint study and multi-component content analysis method of Ganoderma lucidum green tea capsule according to claim 1, characterized in that: In the step S101, the mass concentrations of the prepared epigallocatechin, catechin, ganoderic acid C2, ganoderic acid G, ganoderic acid A, ganoderic acid D, ganoderic acid D and ganoderic acid F are 0.51, 2.39, 0.10, 0.12, 0.34, 0.13, 0.25 and 0.13 mg / mL respectively.

3. The HPLC fingerprint study and multi-component content analysis method of Ganoderma lucidum green tea capsule according to claim 1, characterized in that: The chromatographic conditions in steps S202 and S405 are as follows: chromatographic column: WondaSil C18-WR (250 mm×4.6 mm, 5 μm); mobile phase: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), low-pressure gradient elution, flow rate: 1.0 mL·min-1; column temperature: 30°C; detection wavelength: 254 nm; injection volume: 10 μL.

4. The HPLC fingerprint study and multi-component content analysis method of Ganoderma lucidum green tea capsule according to claim 1, characterized in that: In step S301, when the squared Euclidean distance is 5, the 10 batches of samples can be divided into 2 categories. Samples 1 and 2 obtained after extraction from medicinal materials from the same supplier are grouped into the first category, and samples 3-10 obtained after extraction from medicinal materials from another supplier are grouped into the second category.

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