Method for constructing high performance liquid characteristic chromatogram of ganoderma capense and application of method

Abstract

The invention provides a method for constructing a ganoderma capense high performance liquid characteristic chromatogram and application thereof, and belongs to the technical field of traditional Chinese medicine detection.The method comprises the steps that a certain weight of a ganoderma capense sample is extracted with a methanol solution, and a ganoderma capense extracting solution is obtained; centrifuging the extracting solution, evaporating to dryness, and dissolving with a methanol solution to obtain a test solution; the method comprises the following steps: dissolving uracil, uridine, hypoxanthine, xanthine, inosine, guanosine and adenosine with a certain weight by using a methanol solution to obtain a reference substance solution; respectively taking a proper amount of the test solution and the reference solution, injecting into a high performance chromatograph, recording a chromatogram, analyzing the chromatogram to obtain a ganoderma capense high performance liquid characteristic spectrum, and calculating the relative retention time and the relative peak area of each common peak; the invention establishes a high performance liquid characteristic chromatogram method for detecting ganoderma capense, can realize primordial identification and quality detection, and has the advantages of simplicity in operation, high precision and good stability and repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and in particular to a method for constructing a high-performance liquid phase characteristic spectrum of Ganoderma lucidum and an application thereof. Background Art

[0002] Ganoderma lucidum (Ganoderma lucidum) is the dried fruiting body of the fungus Ganoderma lucidum, a subgenus of the Polyporaceae family. It is also known as Ganoderma lucidum or Ganoderma lucidum. Recent studies have found that Ganoderma lucidum has a composition similar to that of Ganoderma lucidum, including polysaccharides, triterpenes, nucleosides, alkaloids, and sterols, among other active ingredients, with anti-tumor, immunomodulatory, and antioxidant properties. In the field of traditional Chinese medicine (TCM), nucleosides are particularly valuable and are often used as an important indicator for evaluating the quality of TCMs such as Cordyceps sinensis. The TCM characteristic map is a comprehensive and quantifiable identification method that comprehensively reflects the chemical composition characteristics of TCMs, providing an objective and standardized evaluation method for quality control. However, existing research has largely focused on detecting polysaccharide components in Ganoderma lucidum. For example, Chinese patent CN116908124A discloses a method for evaluating the quality of Ganoderma lucidum. This method, which measures the polysaccharide components in Ganoderma lucidum (UV-visible spectrophotometry), is used to assess the quality of Ganoderma lucidum. However, this method cannot distinguish the types and proportions of nucleoside substances and is susceptible to interference. Furthermore, the prior art "Study on HPLC Characteristic Spectra of Ganoderma Lucidum and Its Application in Origin Traceability" discloses an HPLC characteristic spectrum based on ganoderic acid F as a reference peak. However, its characteristic peak does not contain nucleoside components such as uracil, uridine, and adenosine, and it does not involve base identification, thus failing to solve the problem of distinguishing Ganoderma lucidum from other Ganoderma species. Existing Ganoderma lucidum quality evaluation methods lack species-specific parameters. For example, the types and contents of chemical components of Ganoderma lucidum are highly similar to those of Ganoderma lucidum, such as Ganoderma lucidum and Ganoderma linguisticum. Currently, there is no reliable indicator to distinguish Ganoderma lucidum from other Ganoderma medicinal materials. Therefore, there is an urgent need for a method with simple operation, high precision, good stability and repeatability for the quality evaluation and origin identification of Ganoderma lucidum. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides a method for constructing a high-performance liquid chromatography characteristic spectrum of Ganoderma lucidum, which has the characteristics of simple operation, high precision, good stability and repeatability.

[0004] The present invention adopts the following technical solutions:

[0005] In a first aspect, the present invention provides a method for constructing a high-performance liquid chromatography characteristic spectrum of Ganoderma lucidum, comprising:

[0006] S1, extracting a certain weight of Ganoderma lucidum sample with methanol solution to obtain Ganoderma lucidum extract; the extract is centrifuged, evaporated to dryness, and dissolved in methanol solution to serve as a test solution;

[0007] S2, take a certain weight of uracil, uridine, hypoxanthine, xanthine, inosine, guanosine, and adenosine, dissolve them in methanol solution, and use them as reference solution;

[0008] S3, respectively taking appropriate amounts of the test solution and the reference solution and injecting them into a high performance chromatograph for analysis, recording the chromatogram, analyzing the chromatogram, obtaining a high performance liquid chromatography characteristic spectrum of Ganoderma lucidum and calculating the relative retention time and relative peak area of ​​each common peak;

[0009] The chromatographic conditions of the high performance liquid phase are:

[0010] A Zorbax SB-Aq column was selected; acetonitrile-formic acid solution was selected as the mobile phase, and the detection wavelength was 240-280 nm; gradient elution was performed; the gradient elution conditions were as follows: acetonitrile was used as mobile phase A, 0-10 min, the volume of mobile phase A increased from 0.5% to 2%; 10 min-15 min, the volume of mobile phase A increased from 2% to 8%; 15 min-30 min, the volume of mobile phase A increased from 8% to 9%; 30-35 min, the volume of mobile phase A decreased from 9% to 0.5%.

[0011] Furthermore, the flow rate of the method is 0.4-1.0 mL·min -1 .

[0012] Furthermore, the flow rate of the method is 0.6 mL·min -1 .

[0013] Furthermore, the column temperature of the method is 25-40 °C.

[0014] Furthermore, the column temperature of the method is 35°C.

[0015] Furthermore, the injection volume of the method described is 5-20 μL.

[0016] Furthermore, the injection volume of the method described is 20 μL.

[0017] Furthermore, the concentration of formic acid in the mobile phase acetonitrile-formic acid solution is 0.01-0.2%.

[0018] Furthermore, the concentration of formic acid in the mobile phase acetonitrile-formic acid solution is 0.025%.

[0019] Furthermore, the detection wavelength is 260 nm.

[0020] Furthermore, the volume concentration of the methanol solution in the preparation of the test solution in step S1 is 10-30%.

[0021] Furthermore, the volume concentration of the methanol solution in the preparation of the test solution in step S1 is 20%.

[0022] Furthermore, in step S1, the extraction is performed by ultrasonic extraction, and the ultrasonic extraction time is 30-75 min.

[0023] Furthermore, the ultrasonic extraction time is 65 min.

[0024] Furthermore, the concentrations of the components in the control solution in step S2 are: uracil 0.014 mg·mL -1 , uridine 0.025 mg·mL -1 , hypoxanthine 0.010 mg·mL -1 , xanthine 0.010 mg·mL -1 , inosine 0.010 mg·mL -1 , guanosine 0.010 mg·mL -1 , adenosine 0.010 mg·mL -1 .

[0025] Furthermore, the step S3 establishes a generated standard characteristic spectrum, and a total of 7 peaks are determined. Peak 2 (uridine) chromatographic peak is selected as the reference peak S, and the relative retention time is calculated. The 7 characteristic peaks are as follows according to the relative retention time: Peak 1 is uracil, with a relative retention time of 0.819, Peak 2 is uridine, with a relative retention time of 1.000, Peak 3 is hypoxanthine, with a relative retention time of 1.409, Peak 4 is xanthine with a relative retention time of 1.477, Peak 5 is inosine, with a relative retention time of 1.528, Peak 6 is guanosine, with a relative retention time of 1.632, and Peak 7 is adenosine, with a relative retention time of 2.002. The relative retention time should be within ±5% of the specified value.

[0026] In a second aspect, the present invention also provides the application of a method for constructing a high-performance liquid chromatography characteristic spectrum of Ganoderma lucidum in the identification of the origin of Ganoderma lucidum medicinal materials and their processed products.

[0027] Furthermore, in the application of the origin identification, the test sample solution is measured according to the method of the high-performance liquid chromatography characteristic spectrum of Ganoderma lucidum constructed as mentioned above, and the obtained high-performance liquid chromatography characteristic spectrum has 7 characteristic peaks. Peak 2 (uridine) chromatographic peak is selected as the reference peak S, and the relative retention time is calculated. The relative retention times of peaks 1 to 7 are 0.819, 1.000, 1.409, 1.477, 1.528, 1.632, and 2.002, respectively. The relative retention time should be within ±5% of the specified value, and the peak area ratio of peak 7 to peak 2 is less than 0.3, which means it is Ganoderma lucidum medicinal material or its processed product.

[0028] In a third aspect, the present invention also provides the application of a method for constructing a high-performance liquid chromatography characteristic spectrum of Ganoderma lucidum in the quality inspection of Ganoderma lucidum medicinal materials and their processed products.

[0029] Furthermore, the quality detection method uses uracil and uridine as quantitative indicators for quality evaluation, and its steps include: preparing a reference solution, preparing a test solution, accurately aspirating the reference solution and the test solution respectively, injecting them into a high-performance liquid chromatography according to the method of the high-performance liquid phase characteristic spectrum of the thin tree ganoderma constructed above, and performing content determination.

[0030] Furthermore, the reference solution is prepared by taking a certain weight of uracil and uridine reference substances and dissolving them in methanol solution to obtain the reference solution.

[0031] Furthermore, the concentrations of the components in the reference solution are: uracil 14.25 μg·mL -1 , uridine 25.30 μg·mL -1 .

[0032] Furthermore, the preparation of the test solution is the same as S1 in the above characteristic spectrum method.

[0033] Furthermore, the chromatographic conditions of the quality detection method are the same as those of the above-mentioned characteristic spectrum method.

[0034] The present invention has the following beneficial effects:

[0035] (1) The present invention provides a method for constructing a high-performance liquid chromatography characteristic spectrum of Ganoderma lucidum, which uses an acetonitrile-0.025% formic acid solution gradient elution system combined with a Zorbax SB-Aq chromatographic column to achieve efficient separation of seven nucleoside components with significant polarity differences in Ganoderma lucidum. The method has high sensitivity and short analysis time.

[0036] (2) The method of the HPLC characteristic spectrum of Ganoderma lucidum in the present invention is applied to the origin identification of Ganoderma lucidum medicinal materials and their processed products. The HPLC characteristic spectrum has 7 characteristic peaks, and the peak 2 (uridine) chromatographic peak is selected as the reference peak S. The relative retention time is calculated. The relative retention times of peaks 1 to 7 are 0.819, 1.000, 1.409, 1.477, 1.528, 1.632, and 2.002, respectively. The relative retention time should be within ±5% of the specified value, and the peak area ratio of peak 7 to peak 2 is less than 0.3, which means it is Ganoderma lucidum medicinal materials or their processed products. This significantly improves the species identification accuracy of Ganoderma lucidum medicinal materials and solves the problem of insufficient specificity of traditional single-index detection methods.

[0037] (3) The present invention also provides a method for constructing a high-performance liquid chromatography characteristic spectrum of Ganoderma lucidum for use in the quality inspection of Ganoderma lucidum medicinal materials and their processed products. Uracil and uridine are used as quantitative indicators for quality evaluation. After methodological verification, the linear relationship, precision, stability, repeatability and sample recovery rate are good, which fills the technical gap in the quantitative analysis of nucleoside components of Ganoderma lucidum and provides a scientific and comprehensive evaluation system for the quality control of Ganoderma lucidum. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is an investigation of the chromatographic columns in the examples; among them, S1~S2 are Waters Corporation columns, and S3~S4 are Zorbax SB-Aq columns.

[0039] Figure 2 The mobile phase in the embodiment is investigated; among them, S1~S7 are methanol-0.02% formic acid, methanol-0.05% formic acid, methanol-0.075 formic acid, methanol-0.1% formic acid, acetonitrile-0.025% formic acid, acetonitrile-0.05% formic acid, acetonitrile-0.1% formic acid system respectively; a~h are uracil, uridine, adenine, hypoxanthine, xanthine, inosine, guanosine, and adenosine respectively.

[0040] Figure 3 This is a flow rate investigation in the examples; among them, S1, S3, S5, S7, and S9 are mixed controls; S2, S4, S6, S8, and S10 are Ganoderma lucidum.

[0041] Figure 4 This is the column temperature investigation in the examples.

[0042] Figure 5 This is an investigation of the detection wavelength in the examples.

[0043] Figure 6 This is the extraction solvent used in the examples.

[0044] Figure 7 This is an investigation of the extraction time in the examples.

[0045] Figure 8 The HPLC characteristic spectra of 23 batches of Ganoderma lucidum.

[0046] Figure 9 This is a comparison chart of the HPLC characteristic spectra of 7 species of Ganoderma.

[0047] Figure 10 This is the dendrogram of cluster analysis (HCA) of 47 batches of Ganoderma samples.

[0048] Figure 11 The two-dimensional scatter plot of principal component analysis (PCA) scores of 47 batches of Ganoderma samples.

[0049] Figure 12The scatter plots are for 47 batches of Ganoderma samples using orthogonal partial least squares discriminant analysis (OPLS-DA).

[0050] Figure 13 Variable importance projection (VIP) analysis for 47 batches of Ganoderma samples; F1-F7 represent peaks 1-7.

[0051] Figure 14 This is a stacked bar chart of the peak areas of 23 batches of Ganoderma lucidum.

[0052] Figure 15 This is a cumulative graph of the peak area percentages of 23 batches of Ganoderma lucidum.

[0053] Figure 16 The peak areas (A) and peak area percentage stacking plots (B) of the comparison spectra of seven Ganoderma species.

[0054] Figure 17 HPLC spectrum of the mixed reference solution (a) and the HPLC spectrum of the Ganoderma lucidum sample solution (b). DETAILED DESCRIPTION

[0055] The following will be combined with the accompanying drawings and embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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 making any creative efforts shall fall within the scope of protection of the present invention.

[0056] To further illustrate the present invention, the following examples provide a detailed description of a method for constructing a high-performance liquid chromatography (HPLC) characteristic spectrum of Ganoderma lucidum provided by the present invention and its application. Where specific techniques or conditions are not specified in the examples, the product specifications were followed. Where the manufacturer of reagents or instruments is not specified, all are conventional products available through authorized distributors.

[0057] Example

[0058] 1. Instruments and reagents

[0059] 1.1 Instrument

[0060] HP1200 and HP1260 high-performance liquid chromatographs (Agilent, USA, equipped with a diode array detector, autosampler, and column oven); MS204S (d = 0.1 mg) electronic balance (Mettle Teledo, Switzerland); XPR36 (d = 0.001 mg) electronic balance (Mettle Teledo, Switzerland); LS2200C SCS (d = 0.01 g) electronic balance (Mettle Teledo, Switzerland); KQ-400KDE high-efficiency CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); TDL-320 medical centrifuge (Shandong Bio Medical Technology Co., Ltd.); HE53 moisture analyzer (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); Agilent Zorbax SB-Aq chromatographic column (column length, 25 cm, column inner diameter, 4.6 mm, particle size, 5 μm); Waters Corporation chromatography column (column length: 25 cm, column inner diameter: 4.6 mm, particle size: 5 μm).

[0061] 1.2 Reagents and test drugs

[0062] Acetonitrile was chromatographically pure (Merck, Germany), water was ultrapure water, and other reagents were of analytical grade; uracil reference substance (batch number: 100469-202404), uridine reference substance (batch number: 110887-202305), adenine reference substance (batch number: 110886-202404), hypoxanthine (batch number: 140661-202005), xanthine reference substance (batch number: 140662-200802), inosine reference substance (batch number: 110887-202305), and urea-stimulated naphthalene (batch number: 110886-202404) were used as the reference. : 140669-202007), guanosine reference substance (batch number: 111977-202202), and adenosine reference substance (batch number: 110829-202204) were all purchased from the China Food and Drug Inspection Institute; the sources and numbers of the medicinal materials or decoction pieces of Ganoderma lucidum, Ganoderma lucidum (red ganoderma, purple ganoderma), Ganoderma lucidum without sessile, Ganoderma lucidum flat-capped, Ganoderma lucidum tree tongue, and Ganoderma lucidum black are shown in Table 1. They were identified as Ganoderma lucidum thin-capped (Ganoderma lucidum thin-capped) by Huang Yuechun, chief Chinese medicine pharmacist of the First Affiliated Hospital of Guangzhou University of Chinese Medicine, respectively. Fruiting bodies of Ganoderma capense (Lioyd) Teng, G.lucidum (Leyss. ex Fr.)Karst, G.sinense Zhao, Xu et Zhang, G.daipingshanense JDZhao, G.applanatum (Pers.) Pat, G.applanatum (Pers.ex Gray) Pat, and G. atrum.

[0063] Table 1 Sources of Ganoderma lucidum and other Ganoderma medicinal materials

[0064] Serial number name Specification Origin Collection time Serial number name Specification Origin Collection time S1 Boshuzhi medicinal materials Hainan 2023.1 S24 Chizhi medicinal materials Hainan 2023.1 S2 Boshuzhi medicinal materials Hainan 2023.1 S25 Chizhi medicinal materials Hainan 2023.1 S3 Boshuzhi medicinal materials Hainan 2023.1 S26 Chizhi medicinal materials Hainan 2023.1 S4 Boshuzhi medicinal materials Hainan 2023.1 S27 Chizhi medicinal materials Hainan 2023.1 S5 Boshuzhi medicinal materials Hainan 2023.1 S28 Chizhi Herbal Pieces Hainan 2023.1 S6 Boshuzhi medicinal materials Hainan 2023.1 S29 Chizhi Herbal Pieces Hainan 2023.1 S7 Boshuzhi medicinal materials Hainan 2023.1 S30 Chizhi Herbal Pieces Hainan 2023.1 S8 Boshuzhi medicinal materials Hainan 2023.1 S31 Chizhi medicinal pieces Hainan 2023.1 S9 Boshuzhi medicinal materials Hainan 2023.1 S32 Chizhi Herbal Pieces Guangxi 2023.1 S10 Boshuzhi medicinal materials Hainan 2023.1 S33 Ganoderma lucidum medicinal materials Jilin 2023.1 S11 Boshuzhi medicinal materials Hainan 2023.6 S34 Ganoderma lucidum medicinal materials Jilin 2023.1 S12 Boshuzhi medicinal materials Hainan 2023.6 S35 Ganoderma lucidum medicinal materials Jilin 2023.1 S13 Boshuzhi medicinal materials Jilin 2025.2 S36 Ganoderma lucidum medicinal materials Jilin 2023.1 S14 Boshuzhi medicinal materials Guangxi 2025.2 S37 Ganoderma lucidum Herbal Pieces Hainan 2023.1 S15 Boshuzhi medicinal materials Guangxi 2025.2 S38 Ganoderma lucidum Herbal Pieces Hainan 2023.1 S16 Boshuzhi Herbal Pieces Hainan 2023.1 S39 Ganoderma lucidum Herbal Pieces Hainan 2023.1 S17 Boshuzhi Herbal Pieces Hainan 2023.1 S40 Ganoderma lucidum Herbal Pieces Hainan 2023.1 S18 Boshuzhi Herbal Pieces Hainan 2023.1 S41 Sessile Ganoderma lucidum medicinal materials Hainan 2023.6 S19 Boshuzhi Herbal Pieces Hainan 2023.1 S42 Sessile Ganoderma lucidum medicinal materials Hainan 2023.7 S20 Boshuzhi Herbal Pieces Hainan 2023.1 S43 Flat-capped Ganoderma medicinal materials Hainan 2023.7 S21 Boshuzhi Herbal Pieces Hainan 2023.1 S44 Flat-capped Ganoderma medicinal materials Hainan 2023.7 S22 Boshuzhi Herbal Pieces Guangdong 2023.1 S45 Ganoderma lucidum medicinal materials Jilin 2025.2 S23 Boshuzhi Herbal Pieces Guangdong 2023.1 S46 Ganoderma lucidum medicinal materials Jilin 2025.2 - - - - - S47 Black Ganoderma medicinal materials Hainan 2023.6

[0065] Note: “-” means there is no such item.

[0066] 2. Establishment of feature map method

[0067] 2.1 Chromatographic conditions and test sample preparation

[0068] 2.1.1 Column selection

[0069] Under the above chromatographic conditions, two columns were compared: an Agilent Zorbax SB-Aq column (column length: 25 cm, column inner diameter: 4.6 mm, particle size: 5 μm) and a Waters Corporation column of the same specification (column length: 25 cm, column inner diameter: 4.6 mm, particle size: 5 μm). Given the high water solubility of nucleosides, the results showed that using the former as a tail-capped column prolonged the retention time of the target components and improved peak resolution ( Figure 1 ), so the Agilent Zorbax SB-Aq column was selected.

[0070] 2.1.2 Selection of mobile phase system

[0071] According to the above chromatographic conditions, the methanol-formic acid solution and acetonitrile-formic acid solution systems were investigated. Figure 2 As shown in the results, the relative retention times of some chromatographic peaks (especially adenine) in the mixed control solution under different formic acid concentration conditions shifted significantly, and an acetonitrile-formic acid solution system with better separation was preliminarily screened. Further comparison of the spectra under different acidity conditions showed that the acetonitrile-0.025% formic acid system was better.

[0072] 2.1.3 Flow rate selection

[0073] According to the above chromatographic conditions, the flow rates were 0.4, 0.5, 0.6, 0.8, and 1.0 mL·min -1 Effects on the separation of nucleosides from Ganoderma lucidum, such as Figure 3 As shown, the flow rate is 0.6 mL min -1 The separation effect is good, so the flow rate is determined to be 0.6 mL min -1 .

[0074] 2.1.4 Column temperature selection

[0075] Further optimize the HPLC conditions and select different column temperatures for separation (25℃, 30℃, 35℃, 40℃). Figure 4As shown in the figure, when the column temperature was set at 35℃, the separation effect of nucleosides in Ganoderma lucidum was better.

[0076] 2.1.5 Detection wavelength investigation

[0077] According to the above chromatographic conditions, the detection was performed at wavelengths of 240 nm, 250 nm, 260 nm, 270 nm, and 280 nm. Figure 5 As shown in the figure, when the detection wavelength is 260 nm, the separation is good and the peak shape is clear, so the detection wavelength is selected as 260 nm.

[0078] 2.2 Preparation and investigation of test samples

[0079] 2.2.1 Investigation of extraction solvents

[0080] Take 0.5 g of the test sample (S3), accurately weigh it, and place it in a stoppered conical flask. Add 50 mL of 10% methanol solution, 20% methanol solution, and 30% methanol solution respectively. Ultrasonic extraction is carried out for 60 minutes. Take it out, cool it, and centrifuge it (3800 r·min). -1 , 5 min), the supernatant was evaporated to dryness in a water bath, and the methanol solution was diluted to 5 mL. The results showed that when 20% (v / v) methanol solution was used as the extraction solvent, the separation effect of each chromatographic peak and the response value were relatively good ( Figure 6 ), so the extraction solvent for the test sample was 20% methanol solution.

[0081] 2.2.2 Extraction time investigation

[0082] Take 0.5 g of the test sample (S3), accurately weigh it, place it in a stoppered conical flask, add 50 mL of 20% methanol solution, and ultrasonically treat it. The extraction time of the test sample is 30 minutes, 45 minutes, 60 minutes, and 75 minutes respectively. Let it cool and centrifuge it (3800 r·min -1 , 5 min), the supernatant was evaporated to dryness in a water bath, and the methanol solution was diluted to 5 mL. The results showed that the extraction time was 60 minutes and the extraction was sufficient ( Figure 7 ), so the extraction time of the test sample was determined to be 60 minutes.

[0083] 2.2.3 Summary

[0084] Based on the above results, the preparation method of the test sample was determined as follows: 0.5 g of the test sample was accurately weighed and placed in a stoppered conical flask, 50 mL of 20% methanol solution was added, and ultrasonic extraction was performed for 60 minutes. The sample was taken out, cooled, and centrifuged (3800 r·min). -1 , 5 min), evaporate the supernatant in a water bath to dryness, and dilute the volume of methanol solution to 5 mL to obtain the product.

[0085] 2.3 Methodological Review

[0086] 2.3.1 Common Peak Determination

[0087] Each batch of test sample solution was injected and tested according to the above chromatographic conditions, and the chromatograms were recorded. Data analysis was performed using the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition). Using S1 as the reference spectrum and a time window width of 0.1 min, the mean method was used for multi-point correction and full-spectrum peak matching to generate fingerprint overlap plots and a reference fingerprint (R). The characteristic chromatograms of 23 batches of Ganoderma lucidum revealed seven common peaks with stable peak shapes. Through positioning with reference materials and UV spectral comparison, these seven characteristic peaks were identified: peak 1 for uracil, peak 2 for uridine, peak 3 for hypoxanthine, peak 4 for xanthine, peak 5 for inosine, peak 6 for guanosine, and peak 7 for adenosine. Nine batches of Ganoderma lucidum, eight batches of Ganoderma sinensis, two batches of Ganoderma lucidum sessile, two batches of Ganoderma lucidum flat-capped, two batches of Ganoderma lucidum tangerinae, and one batch of Ganoderma lucidum nigra all showed seven characteristic peaks, but the peak areas of some of these peaks were particularly small. The characteristic spectra and comparison spectra of 15 batches of Ganoderma lucidum medicinal materials (S1-S15) and 8 batches of Ganoderma lucidum decoction pieces (S16-S23) are shown in Figure 8 , the comparison and superposition of the atlas of Ganoderma lucidum and other Ganoderma species is shown in Figure 9 Similarity software evaluation showed that within the Ganoderma lucidum medicinal material group, except for sample S15, which had a particularly large relative ratio of uracil peak areas and a similarity of less than 0.7, the other similarities were greater than 0.9; the similarity within the Ganoderma lucidum decoction piece group was greater than 0.9; and the similarity between Ganoderma lucidum and the other six Ganoderma species was less than 0.9.

[0088] 2.3.2 Precision test

[0089] Accurately pipette 20 μL of the same test solution (S3) and inject it six times. Chromatographic peaks (excluding adenosine) with an area exceeding 5% of the total peak area were selected for analysis. The uridine peak was used as the reference peak (S), and the consistency of the relative retention times and peak areas of each chromatographic peak was calculated. The results showed that the RSDs of the relative retention times of uracil, hypoxanthine, xanthine, inosine, and guanosine were all less than 0.5%, and the RSDs of the relative peak areas were all less than 1.2%, indicating good instrument precision.

[0090] 2.3.3 Stability test

[0091] Accurately pipette 20 μL of the same test solution (S3) and inject at 0, 4, 8, 12, 18, and 24 hours. Using the uridine peak as the reference peak (S), the consistency of the relative retention times and relative peak areas of each chromatographic peak was calculated. The results showed that the RSDs of the relative retention times and relative peak areas of the five chromatographic peaks, including uracil, were less than 0.3% and 1.1%, respectively, indicating that the test solution was stable within 24 hours.

[0092] 2.3.4 Repeatability test

[0093] Six aliquots of the same sample (S3) were prepared and injected according to the same method. Using the uridine peak as the reference peak (S), the consistency of the relative retention times and peak areas of each chromatographic peak was calculated. The results showed that the RSDs for the relative retention times and relative peak areas of the five chromatographic peaks, including uracil, were less than 0.5% and 0.9%, respectively, demonstrating good reproducibility of the method.

[0094] 2.4 Chemical pattern recognition

[0095] 2.4.1 HCA analysis

[0096] The peak area data of the 7 common peaks of the above 47 batches of samples were imported into SIMCA14.1 for cluster analysis. The results are shown in Figure 10 The 47 samples can be clustered into three categories: most of the thin tree ganoderma are clustered into category I, the purple ganoderma are clustered into category II, and the red ganoderma are clustered into category III. The sessile red ganoderma and the flat-capped ganoderma are clustered together with the red ganoderma, and the tree tongue and black ganoderma are basically clustered together with the purple ganoderma.

[0097] 2.4.2 PCA analysis

[0098] The data statistical analysis software SIMCA 14.1 was used to perform unsupervised principal component analysis (PCA) with the peak areas of the seven common peaks as variables. The model quality parameter R 2 X=0.823, Q 2 =0.603, where R 2 X represents the cumulative explanation rate of the model in the X-axis direction, that is, the square of the percentage of original data information retained in the X-axis direction; Q 2 Represents the cumulative prediction rate of the model. R 2 X is greater than 0.8, Q 2 The value is greater than 0.6, indicating that the model has good stability and predictive power. The 47 samples can be divided into three categories: thin tree ganoderma is clustered into category I, red ganoderma is clustered into category II, and purple ganoderma is clustered into category III. Sessile red ganoderma and flat-capped ganoderma are clustered together with red ganoderma, and tree tongue and black ganoderma are basically clustered together with purple ganoderma, which is basically consistent with the HCA results. Figure 11 .

[0099] 2.4.3 OPLS-DA analysis

[0100] To further observe the differences between different samples, the 47 samples were divided into 3 groups based on PCA and supervised OPLS-DA analysis was performed. The results are shown in Figure 12 , related model parameters (R 2 X=0.887, R 2 Y=0.732,Q 2 =0.690; R 2 X and Q2 The meaning is the same as PCA analysis, R 2 Y represents the cumulative explanation rate of the model in the Y-axis direction, that is, the square of the percentage of original data information retained in the Y-axis direction). 2 Above 0.6, it indicates that the model has good stability and predictive power and the results are reliable.

[0101] 2.4.4 VIP Analysis

[0102] Based on OPLS-DA, variable projection importance (VIP) analysis was performed to screen components with VIP values ​​greater than 1.0. The results showed that the VIP value of peak 7 (adenosine) was greater than 1.0. Figure 13 , indicating that adenosine is the main differential nucleoside component between different types of Ganoderma lucidum, that is, a significant differential quality marker.

[0103] 2.4.5 Characteristic Peak Area Analysis

[0104] Analysis of the seven characteristic peak areas of 47 samples showed that the peak area of ​​most Ganoderma lucidum samples showed uridine as the first strongest peak, uracil as the second strongest peak, and adenosine as the weakest peak. However, the other six Ganoderma species, such as Ganoderma lucidum, showed uridine and adenosine as the second strongest peak. The peak areas and peak area percentages of multiple batches of each variety were compared using a bar chart (see Figure 14 、 Figure 15 、 Figure 16 ), the results showed that the total peak area of ​​the common peaks of Ganoderma lucidum, Ganoderma rubrum, and Ganoderma lucidum was significantly larger than that of other Ganoderma species, and the peak areas of different samples varied greatly. Among the main peaks, the peak areas of uracil, uracil, and adenosine were the most significantly different. Peak 2 (uridine chromatographic peak) was selected as the reference peak (S), and the relative peak areas of the characteristic peaks of each sample of the seven Ganoderma species were calculated (see Table 3). The relative retention times of the 7 characteristic peaks of 23 batches of Ganoderma lucidum were: peak 1 (0.819), peak 2 (1.000), peak 3 (1.409), peak 4 (1.477), peak 5 (1.528), peak 6 (1.632), and peak 7 (2.002), with RSD less than 0.15%, indicating that the chromatographic conditions and methods were feasible. Except for peaks 1 and 2, which had large differences in the relative peak areas of the common peaks of Ganoderma lucidum, the other relative differences were relatively small. However, the difference in the adenosine to uridine peak area ratio between Ganoderma lucidum and the other six species of Ganoderma lucidum is the most significant. The ratio of Ganoderma lucidum is less than 0.3, which is significantly smaller than the ratios of other Ganoderma lucidum (greater than 0.3), and has certain identification significance.

[0105] Table 2 Adenosine to uridine peak area ratios of 47 batches of Ganoderma lucidum characteristic spectra

[0106] Serial number name Relative peak area Serial number name Relative peak area S1 Boshuzhi 0.038 S24 Chizhi 0.590 S2 Boshuzhi 0.188 S25 Chizhi 0.312 S3 Boshuzhi 0.080 S26 Chizhi 0.410 S4 Boshuzhi 0.150 S27 Chizhi 0.415 S5 Boshuzhi 0.156 S28 Chizhi 0.717 S6 Boshuzhi 0.208 S29 Chizhi 0.484 S7 Boshuzhi 0.062 S30 Chizhi 0.535 S8 Boshuzhi 0.041 S31 Chizhi 0.544 S9 Boshuzhi 0.041 S32 Chizhi 0.503 S10 Boshuzhi 0.099 S33 Ganoderma lucidum 0.402 S11 Boshuzhi 0.105 S34 Ganoderma lucidum 0.352 S12 Boshuzhi 0.082 S35 Ganoderma lucidum 0.399 S13 Boshuzhi 0.282 S36 Ganoderma lucidum 0.341 S14 Boshuzhi 0.113 S37 Ganoderma lucidum 1.325 S15 Boshuzhi 0.074 S38 Ganoderma lucidum 1.218 S16 Boshuzhi 0.112 S39 Ganoderma lucidum 1.532 S17 Boshuzhi 0.118 S40 Ganoderma lucidum 1.273 S18 Boshuzhi 0.114 S41 Sessile Ganoderma lucidum 1.501 S19 Boshuzhi 0.105 S42 Sessile Ganoderma lucidum 1.116 S20 Boshuzhi 0.136 S43 Flat-capped Ganoderma 0.998 S21 Boshuzhi 0.104 S44 Flat-capped Ganoderma 1.676 S22 Boshuzhi 0.176 S45 Ganoderma lucidum 2.116 S23 Boshuzhi 0.161 S46 Ganoderma lucidum 2.267 - - - S47 Black Ganoderma 1.028

[0107] Note: “-” means there is no such item.

[0108] 3 Quality inspection methods

[0109] 3.1 Chromatographic conditions

[0110] The same method as the feature map in 2.1.

[0111] 3.2 Solution preparation

[0112] 3.2.1 Preparation of reference solution

[0113] Take a certain weight of uracil and uridine reference substances and dissolve them in 20% (v / v) methanol solution to prepare the concentration of uracil 14.25 μg mL -1 , uridine 25.30 μg·mL -1 of the reference solution.

[0114] 3.2.2 Preparation of test solution

[0115] The same as the feature map method in 2.2.

[0116] 3.3 Methodological Review

[0117] 3.3.1 Specificity Investigation

[0118] Take uracil and uridine mixed reference solution and test solution respectively and perform determination according to the proposed chromatographic conditions. Figure 17 .

[0119] 3.3.2 Linear relationship investigation

[0120] The stock solution of the mixed reference substance was accurately pipetted to prepare a series of mixed reference substance solutions. Following the proposed chromatographic conditions, a linear regression equation was plotted with the injection volume (μg) as the abscissa (X) and the peak area as the ordinate (Y). The correlation coefficient (r) was calculated. The results showed that for uracil in the range of 0.00570-0.855 μg, the regression equation was Y=6944.3X-12.138 (r=0.9999). For uridine in the range of 0.01012-1.158 μg, the regression equation was Y=3898.9X-14.408 (r=0.9999), indicating a good linear relationship.

[0121] 3.3.3 Precision test

[0122] 20 µL of the same test solution (S3) was accurately pipetted and injected six times. The results showed that the average peak area of ​​uracil was 1829 with an RSD of 0.11%, and the average peak area of ​​uridine was 1523 with an RSD of 0.13%, indicating good instrument precision.

[0123] 3.3.4 Stability test

[0124] Twenty µL of the same test solution (S3) was precisely pipetted and injected at 0, 4, 8, 12, 18, and 24 hours. The results showed that the average peak area for uracil was 1830 with an RSD of 1.31%, and the average peak area for uridine was 1533 with an RSD of 1.23%, indicating good stability of the test solution over 24 hours.

[0125] 3.3.5 Repeatability test

[0126] Six samples (S3) from the same batch were accurately weighed and prepared according to the test solution preparation method. The results showed that the average uracil content was 0.2735 mg·g -1 , RSD was 1.24%, and the average uridine content was 0.4083 mg·g -1 , RSD was 1.31%, indicating that the method had good repeatability.

[0127] 3.3.6 Sample recovery test

[0128] Take 0.25 g of the sample (S3) powder with known content, weigh it accurately, and add uracil reference solution (6.4 uL·mL -1 ), uridine reference solution (10.12 uL·mL -1 ) were added 10 mL each, and then 30 mL of 20% (v / v) methanol solution was accurately added. The weight was weighed and the test solution was prepared according to the method. Six parallel preparations were made and the samples were injected and determined according to the proposed chromatographic conditions. The recovery rates were calculated. The results showed that the average recovery rate of uracil was 97.5% with an RSD of 2.93%, and the average recovery rate of uridine was 97.2% with an RSD of 2.05%, indicating that the recovery rates were good.

[0129] 3.3.7 Sample determination

[0130] According to the proposed chromatographic conditions, each test solution was injected into the liquid chromatograph, the peak area was measured, and the content was calculated by the external standard method. The results (calculated on a dry basis) are shown in Table 3. The contents of uracil, uridine, and the sum of the two in Ganoderma lucidum were 0.015-0.515 mg·g -1 ,0.109-1.230 mg·g -1 ,0.243-1.399 mg·g -1 ; Ganoderma lucidum is 0.001-0.052mg·g -1 ,0.043-0.236 mg·g -1 ,0.045-0.288 mg·g -1 ; Ganoderma lucidum 0.011-0.034 mg·g-1 ,0.286-1.273 mg·g -1 ,0.312-1.285 mg·g -1 Comparing the average uracil and uridine contents in Ganoderma lucidum with those in the other six species of Ganoderma lucidum, the average uracil content in Ganoderma lucidum was higher than that in the other six species of Ganoderma lucidum, indicating that Ganoderma lucidum is different from other Ganoderma lucidum.

[0131] Table 3 Determination results of the content of Ganoderma lucidum (n=2)

[0132] Serial number name Moisture (%) <![CDATA[Uracil (mg·g -1 )]]> <![CDATA[Uridine (mg·g -1 )]]> <![CDATA[Content sum (mg·g -1 )]]> Serial number name Moisture (%) <![CDATA[Uracil (mg·g -1 )]]> <![CDATA[Uridine (mg·g -1 )]]> <![CDATA[Content sum (mg·g -1 ) <!-- 9 -->]]> S1 Boshuzhi 9.88 0.0666 0.8043 0.8709 S24 Chizhi 11.47 0.0017 0.0595 0.0613 S2 Boshuzhi 9.41 0.0809 0.8404 0.9213 S25 Chizhi 11.83 0.0034 0.0473 0.0507 S3 Boshuzhi 9.34 0.3009 0.4473 0.7483 S26 Chizhi 11.57 0.0009 0.0594 0.0604 S4 Boshuzhi 9.16 0.2019 0.4439 0.6459 S27 Chizhi 11.32 0.0018 0.0431 0.0449 S5 Boshuzhi 9.44 0.0699 0.8507 0.9206 S28 Chizhi 9.94 0.0305 0.0806 0.1111 S6 Boshuzhi 9.68 0.0936 0.5266 0.6202 S29 Chizhi 10.86 0.0311 0.0861 0.1172 S7 Boshuzhi 10.33 0.1381 0.6025 0.7406 S30 Chizhi 10.76 0.0331 0.0909 0.1240 S8 Boshuzhi 10.07 0.0637 1.2301 1.2938 S31 Chizhi 10.43 0.0196 0.0798 0.0994 S9 Boshuzhi 9.98 0.2389 1.1596 1.3985 S32 Chizhi 9.35 0.0522 0.2363 0.2884 S10 Boshuzhi 11.05 0.0985 0.7187 0.8173 S33 Ganoderma lucidum 11.22 0.0111 0.7981 0.8092 S11 Boshuzhi 10.2 0.2455 0.4843 0.7298 S34 Ganoderma lucidum 10.49 0.0227 0.6391 0.6617 S12 Boshuzhi 10.49 0.1552 0.5339 0.6891 S35 Ganoderma lucidum 10.66 0.0125 1.2728 1.2853 S13 Boshuzhi 10.91 0.0690 0.4398 0.5088 S36 Ganoderma lucidum 10.59 0.0196 1.2622 1.2818 S14 Boshuzhi 13.59 0.1708 0.3591 0.5299 S37 Ganoderma lucidum 9.44 0.0345 0.3082 0.3427 S15 Boshuzhi 12.07 0.5154 0.2164 0.7318 S38 Ganoderma lucidum 10.87 0.0302 0.3281 0.3583 S16 Boshuzhi 9.44 0.1856 0.4217 0.6073 S39 Ganoderma lucidum 8.98 0.0328 0.2793 0.3121 S17 Boshuzhi 9.38 0.2672 0.3222 0.5894 S40 Ganoderma lucidum 10.00 0.0336 0.2865 0.3201 S18 Boshuzhi 9.40 0.1679 0.4683 0.6362 S41 Sessile Ganoderma lucidum 11.38 0.1051 0.0595 0.1646 S19 Boshuzhi 9.36 0.2914 0.3301 0.6215 S42 Sessile Ganoderma lucidum 12.09 0.0462 0.0539 0.1000 S20 Boshuzhi 9.36 0.1627 0.3583 0.5210 S43 Flat-capped Ganoderma 13.46 0.0218 0.0449 0.0666 S21 Boshuzhi 9.61 0.3049 0.3497 0.6546 S44 Flat-capped Ganoderma 12.32 0.0239 0.0739 0.0977 S22 Boshuzhi 9.22 0.1932 0.1094 0.3026 S45 Ganoderma lucidum 11.91 0.0080 0.0859 0.0939 S23 Boshuzhi 9.26 0.1556 0.2136 0.3691 S46 Ganoderma lucidum 14.43 0.0044 0.1387 0.1431 - - - - - - S47 Black Ganoderma 11.07 0.0395 0.5162 0.5557

[0133] Note: “-” means there is no such item.

[0134] The present invention is further described above with the aid of specific embodiments. However, it should be understood that the specific description herein should not be construed as limiting the essence and scope of the present invention. Various modifications made to the above embodiments by ordinary technicians in this field after reading this specification are all within the scope of protection of the present invention.

Claims

1. A method for constructing a high-performance liquid chromatography characteristic spectrum of Ganoderma lucidum, characterized in that: include: S1, extracting a certain weight of Ganoderma lucidum sample with methanol solution to obtain Ganoderma lucidum extract; the extract is centrifuged, evaporated to dryness, and dissolved in methanol solution to serve as a test solution; S2, take a certain weight of uracil, uridine, hypoxanthine, xanthine, inosine, guanosine, and adenosine, dissolve them in methanol solution, and use them as reference solution; S3, respectively taking appropriate amounts of the test solution and the reference solution and injecting them into a high performance chromatograph for analysis, recording the chromatogram, analyzing the chromatogram, obtaining a high performance liquid chromatography characteristic spectrum of Ganoderma lucidum and calculating the relative retention time and relative peak area of ​​each common peak; The chromatographic conditions of the high performance liquid phase are: A Zorbax SB-Aq column was selected; acetonitrile-formic acid solution was selected as the mobile phase, and the detection wavelength was 240-280 nm; gradient elution was performed; the gradient elution conditions were as follows: acetonitrile was used as mobile phase A, 0-10 min, the volume of mobile phase A increased from 0.5% to 2%; 10 min-15 min, the volume of mobile phase A increased from 2% to 8%; 15 min-30 min, the volume of mobile phase A increased from 8% to 9%; 30-35 min, the volume of mobile phase A decreased from 9% to 0.5%.

2. The method according to claim 1, characterized in that The flow rate of the mobile phase is 0.4-1.0 mL·min -1 .

3. The method according to claim 1, characterized in that The column temperature was 25-40 °C.

4. The method according to claim 1, wherein The injection volume of the test solution and the reference solution into the high performance chromatograph is 5-20 μL.

5. The method for constructing a high-performance liquid chromatography characteristic spectrum of Ganoderma lucidum according to claim 1, characterized in that: The volume concentration of the methanol solution in the preparation of the test solution in step S1 is 10-30%.

6. The method for constructing a high-performance liquid chromatography characteristic spectrum of Ganoderma lucidum according to claim 1, characterized in that: In the step S1, the extraction is performed by ultrasonic extraction, and the ultrasonic extraction time is 30-75 min.

7. The method for constructing a high-performance liquid chromatography characteristic spectrum of Ganoderma lucidum according to claim 1, characterized in that: The step S3 establishes a generated standard characteristic spectrum, and a total of 7 peaks are determined. Peak 2 (uridine) chromatographic peak is selected as the reference peak S, and the relative retention time is calculated. The 7 characteristic peaks are as follows according to the relative retention time: Peak 1 is uracil, with a relative retention time of 0.819, Peak 2 is uridine, with a relative retention time of 1.000, Peak 3 is hypoxanthine, with a relative retention time of 1.409, Peak 4 is xanthine with a relative retention time of 1.477, Peak 5 is inosine, with a relative retention time of 1.528, Peak 6 is guanosine, with a relative retention time of 2.002, and Peak 7 is adenosine, with a relative retention time of 2.

002. The relative retention time should be within ±5% of the specified value.

8. Application of the method for constructing a high-performance liquid chromatography characteristic spectrum of Ganoderma lucidum according to any one of claims 1 to 7 in the identification of the origin of Ganoderma lucidum medicinal materials and their processed products.

9. The use of claim 8 in origin identification, characterized in that The test solution is measured according to the method of the constructed high-performance liquid chromatography characteristic spectrum of Ganoderma lucidum. If the obtained high-performance liquid chromatography characteristic spectrum has 7 characteristic peaks, select Peak 2 (uridine) chromatographic peak as the reference peak S, and calculate the relative retention time. The relative retention times of Peak 1-Peak 7 are 0.819, 1.000, 1.409, 1.477, 1.528, 1.632, and 2.002, respectively. The relative retention time should be within ±5% of the specified value, and the peak area ratio of Peak 7 to Peak 2 is less than 0.3, which means it is Ganoderma lucidum medicinal material or its processed product.

10. Use of the method for constructing a high-performance liquid chromatography characteristic spectrum of Ganoderma lucidum according to any one of claims 1 to 7 in quality inspection of Ganoderma lucidum medicinal materials and their processed products.

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