Lucid ganoderma type identification marker and application thereof

Abstract

The invention relates to the field of traditional Chinese medicines, and discloses a lucid ganoderma type identification marker and application thereof. The invention aims to solve the problem that a system for discriminating wild and non-wild ganoderma lucidum does not exist at present, and provides a marker for discriminating wild and non-wild ganoderma lucidum and application thereof.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine, and in particular to the field of identification and quality evaluation of traditional Chinese medicine. Background Art

[0002] Chinese medicinal materials are the material basis for the prevention and treatment of diseases in traditional Chinese medicine, and are important raw materials for Chinese patent medicines, Chinese medicinal slices, etc. The quality of Chinese medicinal materials is directly related to the physical health and life safety of the general public. According to the different growth patterns of Chinese medicinal materials, they can be divided into wild products and cultivated products. Wild Chinese medicinal materials are the main clinical medicines used by doctors in my country throughout the ages. They are usually affected by various adverse stresses such as drought / floods, high temperature / severe cold, salinity, heavy metals, etc., and have the characteristics of long growth years, rich accumulation of secondary metabolites, and strong efficacy. The origin, properties, quality, efficacy, usage and dosage of high-quality Chinese medicinal materials recorded in the herbal books of all dynasties are based on wild Chinese medicinal materials. The theory of Chinese medicine in my country has developed on the basis of thousands of years of historical experience in the use of wild Chinese medicinal materials and the exploration of their dose-effect relationship.

[0003] Ganoderma lingzhi Sheng H. Wu, Y. Cao & Y. C. Dai belongs to the kingdom of fungi, phylum Basidiomycetes, class Agaricomycetes, order Polyporaces, family Ganodermaceae. It is a famous Chinese herbal medicine. Its application history can be traced back to the Hemudu culture period 6,800 years ago. It has the effects of replenishing qi and calming the mind, relieving cough and relieving asthma. It is often used for restlessness, insomnia and palpitations, cough and asthma caused by lung deficiency, shortness of breath due to consumptive disease, and loss of appetite. Modern pharmacology and clinical practice have further confirmed that Ganoderma has many pharmacological effects such as preventing and treating cardiovascular diseases, protecting the liver, anti-tumor, immunomodulation, anti-aging, and antibacterial.

[0004] Since the 1980s, with the increasing demand for Ganoderma lucidum and the decreasing reserves of wild Ganoderma lucidum resources, Ganoderma lucidum medicinal materials have begun to be widely introduced, domesticated and cultivated. The scale of Ganoderma lucidum cultivation has continued to grow. As of 2023, the planting area of ​​Ganoderma lucidum has exceeded 200,000 mu, and clinical medication and market circulation are mainly cultivated products. Cultivated Ganoderma lucidum has made a huge contribution to meeting the needs of clinical medication. However, since my country's Ganoderma lucidum cultivation mainly draws on the cultivation and management model of crops, there are behaviors such as high-density planting, large amounts of water and fertilizer, the use of pesticides and even plant growth regulators, which has caused significant changes in the appearance and odor of cultivated Ganoderma lucidum, such as the color of the skin becoming lighter, the radial wrinkles on the surface of the cap being significantly reduced, and the texture of the medicinal material becoming softer. Changes in phenotypic traits indicate changes in the intrinsic quality of the medicinal material, which has caused the industry to worry about the quality of cultivated Ganoderma lucidum. Due to trait variation, wild products and cultivated products have been listed separately under the Ganoderma lucidum variety item since the 2000 edition of the "Chinese Pharmacopoeia". It can be seen that the artificial cultivation of Ganoderma lucidum should explore its own laws, and should not blindly copy the cultivation methods of traditional crops. The blind pursuit of yield is very likely to lead to reduced efficacy and quality. At present, there is no identification system for wild and non-wild Ganoderma lucidum. Therefore, it is of great significance to systematically carry out the identification of wild and non-wild Ganoderma lucidum, analyze their quality differences and explore the alternative use of wild and cultivated Chinese medicinal materials. It is also an important scientific exploration to guide the high-quality development of cultivated Chinese medicinal materials and ensure the safety of drug use by the general public. Summary of the invention

[0005] The purpose of the present invention is to solve the problem that there is no type identification system for Ganoderma lucidum at present, and to provide a Ganoderma lucidum type identification marker and its application.

[0006] The basic technical scheme of the present invention is: the Ganoderma lucidum type identification marker comprises any one compound of Ganoderic acid delta and Ganoderic acid H or a combination of the two compounds.

[0007] The inventors used mass spectrometry to measure and analyze the relative contents of various substances in four types of Ganoderma lucidum collected in advance, namely, Ganoderma lucidum cultivated in simulated forest environment (LX), Ganoderma lucidum cultivated in indoor factory environment (SN), Ganoderma lucidum cultivated in greenhouse facility (DP) and wild Ganoderma lucidum (YS). From the experimental results, it can be seen that the relative content of Ganoderic acid delta in wild Ganoderma lucidum is significantly higher than that in Ganoderma lucidum cultivated in simulated forest environment, Ganoderma lucidum cultivated in indoor factory environment and Ganoderma lucidum cultivated in greenhouse facility; the relative content of Ganoderma lucidum cultivated in indoor factory environment is not aggregated but rather dispersed; the relative contents of Ganoderma lucidum cultivated in greenhouse facility and Ganoderma lucidum cultivated in simulated forest environment are relatively aggregated, but are far lower than those of wild Ganoderma lucidum; Ganoderic acid delta The comparison results of the relative content of H showed that the relative content of wild Ganoderma lucidum was significantly lower than that of Ganoderma lucidum cultivated in simulated environment under forest, Ganoderma lucidum cultivated in indoor factory and Ganoderma lucidum cultivated in greenhouse facilities. In addition, Ganoderma lucidum cultivated in greenhouse facilities and indoor factory-cultivated Ganoderma lucidum did not aggregate but were relatively dispersed, while Ganoderma lucidum cultivated in simulated environment under forest was relatively aggregated. In addition, the markers of this scheme have not been reported to be used as markers for identifying Ganoderma types.

[0008] In practical applications, when the type of Ganoderma lucidum cannot be determined, any one of the markers in this scheme or two combined markers can be used to compare the relative contents of each type of Ganoderma lucidum, so as to distinguish wild Ganoderma lucidum from other types of Ganoderma lucidum, thereby providing a strong basis for the current inability to identify the type of wild Ganoderma lucidum, forming a Ganoderma lucidum identification system, and also guiding the high-quality development of cultivated Chinese medicinal materials.

[0009] Furthermore, the identification markers of the types of Ganoderma lucidum also include any one of Lucidone B, Ganoderic acid J, Ganodericacid G, Ganoderic acid C2, Ganoderic acid A, Ganoderic acid B, and any combination of any of the extracts. The present invention also determines the relative contents of several other substances for comparison, and the results show that the relative content comparison results of Lucidone B are as follows: the relative content of wild Ganoderma lucidum is lower than that of Ganoderma lucidum cultivated in simulated environments under forests, Ganoderma lucidum cultivated in indoor factories, and Ganoderma lucidum cultivated in greenhouse facilities, and Ganoderma lucidum cultivated in greenhouse facilities does not aggregate but is more dispersed, while Ganoderma lucidum cultivated in simulated environments under forests and Ganoderma lucidum cultivated in indoor factories are more aggregated; Ganoderic acid J is as follows: the relative content of wild Ganoderma lucidum is significantly higher than that of Ganoderma lucidum cultivated in simulated environments under forests, Ganoderma lucidum cultivated in indoor factories, and Ganoderma lucidum cultivated in greenhouse facilities, and the relative content of Ganoderma lucidum cultivated in indoor factories is not aggregated but is more dispersed, while the relative content of Ganoderma lucidum cultivated in greenhouse facilities and Ganoderma lucidum cultivated in simulated environments under forests is more aggregated, but all are far lower than that of wild Ganoderma lucidum; In the results of the relative content of acidG, the greenhouse-cultivated Ganoderma, the forest-cultivated Ganoderma, the indoor factory-cultivated Ganoderma, and the wild Ganoderma all showed a certain degree of dispersion, but the relative content of the wild Ganoderma was also slightly higher than that of other types; in the test results of the extract, the extract content of the wild Ganoderma was significantly higher than that of other types of Ganoderma, followed by the greenhouse-cultivated Ganoderma, and the difference between the Ganoderma cultivated in the forest-cultivated environment and the indoor factory-cultivated Ganoderma was not much; Ganoderic acid C2, Ganoderic acid A, and Ganoderic acid B all showed that the relative content of the wild Ganoderma was significantly higher than that of the Ganoderma cultivated in the forest-cultivated environment, the indoor factory-cultivated Ganoderma, and the greenhouse-cultivated Ganoderma; the relative content of the indoor factory-cultivated Ganoderma was not concentrated but more dispersed; the relative contents of the greenhouse-cultivated Ganoderma and the forest-cultivated Ganoderma were relatively concentrated, but all were far lower than those of the wild Ganoderma.

[0010] On the basis of the basic scheme, any one, any two, three, four or any combination of the markers in this scheme can be used to compare the relative contents of various types of Ganoderma lucidum, and the accuracy of identification is higher.

[0011] Another basic technical solution of the present invention is a method for identifying the type of Ganoderma lucidum using the Ganoderma lucidum type identification markers of the above solution.

[0012] Further, in the method for identifying the type of Ganoderma lucidum, the contents of Ganoderic acid delta, Ganoderic acid H, Lucidone B, Ganoderic acid J, Ganoderic acid G, Ganoderic acid C2, Ganoderic acid A, and Ganoderic acid B are detected by UHPLC-Q / TOF-MS mass spectrometry. The relative content detection of the present invention adopts UHPLC-Q / TOF-MS mass spectrometry. UHPLC-Q / TOF-MS is an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry technology. This technology combines the strong identification ability of mass spectrometry and the high separation efficiency of ultra-high performance liquid chromatography, and has the advantages of high sensitivity, good selectivity, and fast analysis speed. It is widely used in multiple research fields such as analysis and identification of traditional Chinese medicine components, analysis and identification of in vivo drug components, non-targeted metabolomics, molecular biology, and forensic testing.

[0013] Furthermore, the method for identifying the type of Ganoderma lucidum also obtains a relative content analysis diagram after normalization of the total ion current (TIC). The relative content analysis comparison diagram after normalization of the total ion current (TIC) is usually a box diagram, which can intuitively reflect the relative content position of the Ganoderma lucidum of each sample, so that the type of Ganoderma lucidum of each sample can be obtained very intuitively. Total ion current (TIC) is an important concept in mass spectrometry analysis, which refers to the spectrum of the sum of all ion intensities over time or the number of scans within a selected mass range. The TIC diagram reflects the change of the intensity of all ion signals over time during the mass spectrometry analysis. The generation process of TIC is that in mass spectrometry analysis, after the separated components are ionized by the ion source, the ion flow signals generated by all ions are amplified and correspond to the elution time of the components to form a TIC diagram. These ion signals include ion signals generated after the separated components, residual gases and some carrier gas molecules are ionized. In the mass spectrometer, there is a certain positive correlation between the ion flow intensity and the content of the substance. Generally speaking, the stronger the ion flow intensity, the higher the relative content of the substance in the sample. The relative content of different substances is determined by comparing the height or area of ​​the ion flow peaks corresponding to them. Usually, the intensity of the strongest ion flow peak in the mass spectrum is set as 100%, and the intensity of other ion flow peaks is expressed as a percentage of the intensity of the strongest peak, thereby obtaining the relative content ratio of each substance.

[0014] Furthermore, the method for identifying the type of Ganoderma lucidum also includes a principal component analysis step. The principal components of each type of Ganoderma lucidum can be clustered by using the data after mass spectrometry analysis and principal component analysis to obtain a cluster analysis diagram. Principal component analysis is a cluster analysis, which is an analysis process that groups a collection of physical or abstract objects into multiple classes composed of similar objects. Since the principal components of each type of Ganoderma lucidum are not limited to the above-mentioned markers, hundreds of principal components are subjected to cluster analysis after mass spectrometry analysis. In the obtained analysis diagram, it can be clearly seen that wild Ganoderma lucidum, greenhouse facility-cultivated Ganoderma lucidum, forest-cultivated Ganoderma lucidum, and indoor factory-cultivated Ganoderma lucidum are clustered together separately. Therefore, in the identification method, the type identification of each type of Ganoderma lucidum can be further distinguished by combining cluster analysis.

[0015] Another basic technical solution provided by the present invention is a Ganoderma identification database constructed based on the above-mentioned Ganoderma type identification markers. In practical applications, during the marketing process, the Ganoderma samples of the above-mentioned markers and the measured relative content sample data are accumulated and stored to form a Ganoderma identification database. The larger and more data in the database, the clearer the standards formed, thereby improving the identification accuracy of the Ganoderma identification system.

[0016] Furthermore, the Ganoderma identification database also includes the above-mentioned Ganoderma type identification method. The database also includes the Ganoderma type identification method, thereby more standardized Ganoderma identification system and unified standards.

[0017] Further, the Ganoderma lucidum identification database also includes the content values ​​of any one substance or several substances of Ganoderic acid delta, Ganoderic acid H, Lucidone B, Ganoderic acid J, Ganoderic acid G, Ganoderic acid C2, Ganoderic acid A, Ganoderic acid B, and extracts of each type of Ganoderma lucidum. In the database, the content values ​​of each marker can be identified and stored. When identifying the type of Ganoderma lucidum, it is only necessary to measure the marker content of the sample to be tested to obtain the type of Ganoderma lucidum, which is more convenient and fast. Because in the mass spectrum, there is a certain positive correlation between the ion flow intensity and the content of the substance. Generally speaking, the stronger the ion flow intensity, the higher the relative content of the substance in the sample, which means that the higher the relative content, that is, the higher the absolute content of the substance in the Ganoderma lucidum is determined. Therefore, it is only necessary to measure the absolute content of each marker and store it in the database to form a standard control for the identification of the type of Ganoderma lucidum.

[0018] Further, the compound structural formula of Ganoderic acid delta is , the chemical structure of Ganoderic acid H is , the chemical structure of Lucidone B is , the chemical structure of Ganoderic acid J is The chemical structure of Ganoderic acid G is In order to identify each marker and avoid misunderstanding that the name is inconsistent but it is actually the same compound, the present invention provides the compound structural formula of each marker to further identify each marker.

[0019] Through the research of the present invention, it is found that in the future, the industry will have unified Ganoderma lucidum identification markers and identification methods, as well as a database with determined markers, identification methods and standard sample identification data. Then, on the one hand, the use of Chinese medicinal materials Ganoderma lucidum will be managed in a more standardized manner, and the behavior of passing cultivated Ganoderma lucidum off as wild Ganoderma lucidum will be eliminated; on the other hand, in terms of the medicinal use of Ganoderma lucidum medicinal materials, more suitable types of Ganoderma lucidum can be used for patients in a more targeted manner, thereby avoiding the situation of indiscriminate use of drugs; furthermore, in terms of artificially cultivated Ganoderma lucidum, it can also focus on cultivating varieties with higher content of markers of wild Ganoderma lucidum and lower content of markers of wild Ganoderma lucidum, thereby promoting the quality of artificially cultivated Ganoderma lucidum, standardizing the industry, forming a virtuous cycle of ecology, and making great contributions to the industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparison chart of the relative contents of triterpenoid components of Ganoderma lucidum under the positive ion mode of Example 1 of the present invention. Figure 2 This is a comparison chart of the relative contents of triterpenoid components of Ganoderma lucidum under negative ion mode in Example 1 of the present invention; Figure 3 This is a comparison chart of the effective ingredient content in Ganoderma lucidum according to Example 1 of the present invention; Figure 4 This is a graph showing the principal component analysis scores of Ganoderma lucidum in the positive ion mode according to Example 2 of the present invention; Figure 5 This is a graph showing the principal component analysis scores of Ganoderma lucidum in the negative ion mode of Example 2 of the present invention; Figure 6 This is a graph showing the principal component analysis scores of Ganoderma lucidum in the positive ion mode according to Example 3 of the present invention; Figure 7 This is a graph showing the principal component analysis scores of Ganoderma lucidum in the negative ion mode of Example 3 of the present invention; Figure 8 This is a comparison chart of the relative contents of triterpenoid components of Ganoderma lucidum under the positive ion mode of Example 5 of the present invention. Fig. 9 This is a comparison chart of the relative contents of triterpenoid components of Ganoderma lucidum under the negative ion mode of Example 5 of the present invention; Fig.10 This is a comparison chart of the effective ingredient content in Ganoderma lucidum according to Example 5 of the present invention; Fig.11This is a graph showing the principal component analysis scores of Ganoderma lucidum in the positive ion mode according to Example 6 of the present invention; Fig.12 This is a graph showing the principal component analysis scores of Ganoderma lucidum in the negative ion mode of Example 6 of the present invention. DETAILED DESCRIPTION

[0021] The following is further described in detail through specific implementation methods: Example 1: Analysis of relative contents of triterpenoid components in Ganoderma lucidum 1. Ganoderma lucidum medicinal materials: Ganoderma lucidum samples are shown in Table 1. Each medicinal material sample consists of 3 batches, which are the same type from different regions.

[0022] Table 1 Ganoderma lucidum sample information

[0023] 2. Ganoderma lucidum sample processing After drying to constant weight in an oven at 40-50℃, each Ganoderma lucidum sample was ground at room temperature (60 Hz, 40 s). 50 mg of sample powder was accurately weighed into a 2 mL EP tube using an analytical balance, and two homogenizing grinding beads were added. 500 μL of extract (methanol:acetonitrile:water (2:2:1, v / v / v)) containing an isotope-labeled internal standard was added, and the mixture was vortexed for 30 s. The mixture was homogenized in a homogenizer (35 Hz, 240 s) and then transferred to an ice-water bath for sonication for 5 min. The homogenization and sonication steps were repeated 3 times. Place in -40℃ refrigerator and let stand for 30 min; centrifuge at 4℃, 12000rpm (centrifugal force 13800 (×g), radius 8.6cm) for 15 min, take supernatant; let the supernatant stand in -40℃ refrigerator for 10 min; centrifuge at 4℃, 12000rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 min; take the supernatant through 0.22 μm microporous filter membrane into injection vial, and test on the machine. Take 20 μL supernatant from all samples and mix them into QC samples, and test on the machine.

[0024] 3. Get the original data The present invention uses a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph to perform chromatographic separation of the target compound through a Phenomenex Kinetex C18 (2.1 mm × 100 mm, 2.6 μm) liquid chromatography column. The liquid chromatography phase A is an aqueous phase containing 0.01% acetic acid, and the phase B is isopropanol:acetonitrile (1:1, v / v). The sample plate temperature is 4°C, and the injection volume is 2 μL. The Orbitrap Exploris 120 mass spectrometer can perform primary and secondary mass spectrometry data acquisition under the control of the control software (Xcalibur, version: 4.4, Thermo). The detailed parameters are as follows: Sheath gas flowrate: 50 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 320 ℃, Fullms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, Spray Voltage: 3.8 kV (positive) or -3.4 kV (negative) to obtain the raw data.

[0025] 4. Determination of relative content of triterpenoid components in Ganoderma lucidum 1. Relative content and analysis of triterpenoids in Ganoderma lucidum The raw data were converted into mzXML format using ProteoWizard software and processed by the XCMS program developed based on R language for peak detection, extraction, calibration and integration. The BiotreeDB MS2 database was then used for metabolite annotation, and the cutoff value of the annotation was set to 0.3. The chemical components in the Ganoderma lucidum sample extracts were identified using the BiotreeDB MS2 database, HMDB, Massbank and Chemspider databases. The total ion current (TIC) of each sample was used for normalization and analysis to obtain the following: Figure 1 The comparison chart of the relative contents of triterpenoid components in Ganoderma lucidum under positive ion mode is shown; Figure 2 The figure shows the relative content comparison of triterpenoid components in Ganoderma lucidum under negative ion mode. In the figure, YS is wild Ganoderma lucidum, DP is greenhouse facility cultivated Ganoderma lucidum, LX is cultivated Ganoderma lucidum in simulated environment under forest, and SN is indoor factory cultivated Ganoderma lucidum.

[0026] 2. Analysis results of relative contents of triterpenoids in Ganoderma lucidum Depend on Figure 1 and Figure 2It can be seen that the relative contents of Ganoderic acid C2, Ganoderic acid A, Ganoderic acid_delta, Ganoderic_acid_H, Lucidone_B, Ganoderic_acid_J, Ganoderic acid B and Ganoderic acid G in wild Ganoderma are quite different from those in Ganoderma cultivated by other methods, which can be used as a basis for distinguishing wild Ganoderma from Ganoderma cultivated by other methods.

[0027] 5. Determination of the content of active ingredients in wild Ganoderma lucidum 1. Content of active ingredients in Ganoderma lucidum The contents of extracts, triterpenes and polysaccharides in Ganoderma lucidum and other cultivated varieties were determined according to the methods specified in the 2020 edition of the Chinese Pharmacopoeia.

[0028] 2. Results of the content of active ingredients in Ganoderma lucidum Depend on Figure 3 It can be seen that the contents of extract, triterpenoid components and total polysaccharides in wild Ganoderma lucidum are higher than those in other cultivated varieties, especially the extract is much higher than that in other cultivated varieties.

[0029] Example 2: Principal component analysis of wild Ganoderma lucidum 1. The method of obtaining the Ganoderma lucidum medicinal material, sample processing, and raw data is consistent with step 1 to step 3 of embodiment 1.

[0030] 2. Data Preprocessing and Analysis The raw data of wild Ganoderma lucidum and greenhouse cultivated Ganoderma lucidum (raw data obtained in step 3 of Example 1) were converted into mzXML format using ProteoWizard software and processed by XCMS program developed based on R language for peak detection, extraction, calibration and integration. The BiotreeDB MS2 database was then used for metabolite annotation, and the cutoff value of the annotation was set to 0.3. The chemical components in the Ganoderma lucidum sample extracts were identified using the BiotreeDB MS2 database, HMDB, Massbank and Chemspider databases.

[0031] The total ion current (TIC) of each sample was used for normalization. After obtaining the sorted data, the data were converted to logarithmic (log) and centered (CTR) formats and then subjected to principal component analysis (PCA) using SIMCA software (V15.0.2, Sartorius Stedim Data Analytics AB, Umea, Sweden). Figure 4 As shown in the figure, the principal component analysis score diagram of Ganoderma lucidum in positive ion mode is as follows: Figure 5As shown, the score diagram of the principal component analysis of Ganoderma lucidum under negative ion mode; in the figure, in order to more clearly distinguish the sample numbers at the clustering points of the samples, an enlarged image is placed in the rectangular frame, and the arrow points from the rectangular frame to be enlarged to the enlarged image.

[0032] 3. Principal component analysis of wild Ganoderma lucidum Depend on Figure 4 and Figure 5 It can be seen that the QC samples are clustered together, indicating that the instrument is running stably, the wild Ganoderma lucidum is clustered together, and the greenhouse cultivated Ganoderma lucidum is clustered together, indicating that the wild Ganoderma lucidum can be distinguished from the greenhouse cultivated Ganoderma lucidum through principal component analysis, and the model is successfully established; QC samples are qualified samples used by enterprises in the quality management process to verify whether the products meet the quality standards.

[0033] Example 3: Principal component analysis of various types of Ganoderma lucidum 1. The method of obtaining the Ganoderma lucidum medicinal material, sample processing, and raw data is consistent with step 1 to step 3 of embodiment 1.

[0034] 2. Data Processing and Analysis The raw data of wild Ganoderma lucidum, greenhouse facility-cultivated Ganoderma lucidum, forest-cultivated Ganoderma lucidum, and indoor factory-cultivated Ganoderma lucidum (raw data obtained in step 3 of Example 1) were converted into mzXML format using ProteoWizard software and processed by the XCMS program developed based on R language for peak detection, extraction, calibration, and integration. The BiotreeDB MS2 database was then used for metabolite annotation, and the cutoff value of the annotation was set to 0.3. The chemical components in the Ganoderma lucidum sample extracts were identified using the BiotreeDB MS2 database, HMDB, Massbank, and Chemspider databases.

[0035] The total ion current (TIC) of each sample was used for normalization. After obtaining the sorted data, the data were converted to logarithmic (log) and centered (CTR) formats and then subjected to principal component analysis (PCA) using SIMCA software (V15.0.2, Sartorius Stedim Data Analytics AB, Umea, Sweden). Figure 6 The principal component analysis score diagram of Ganoderma lucidum in positive ion mode is shown in the figure. Figure 7 The figure shows the score diagram of the principal component analysis of Ganoderma lucidum under the negative ion mode; in order to more clearly distinguish the sample numbers at the clustering points of the samples, an enlarged image is placed in the rectangular frame, and the arrow points from the rectangular frame to be enlarged to the enlarged image.

[0036] 3. Principal component analysis of various types of Ganoderma lucidum Depend on Figure 6 and Figure 7It can be seen that the QC samples are clustered together, indicating that the instrument is running stably, the wild Ganoderma lucidum is clustered together, the greenhouse facility-cultivated Ganoderma lucidum is clustered together, the forest-cultivated Ganoderma lucidum is clustered together, and the indoor factory-cultivated Ganoderma lucidum is clustered together, indicating that the principal component analysis method can distinguish wild Ganoderma lucidum from other cultivated types of Ganoderma lucidum.

[0037] Of course, the principal component analysis software can also be used with other software such as SPSS cluster analysis to obtain analysis charts, so as to distinguish various types of Ganoderma lucidum.

[0038] Example 4: Establishment of Ganoderma lucidum identification database The database includes: 1. The method of Embodiment 1 to Embodiment 3; 2. The markers of Examples 1 to 3: Ganoderic acid C2, Ganoderic acid A, Ganoderic acid_delta, Ganoderic_acid_H, Lucidone_B, Ganoderic_acid_J, Ganoderic acid B and Ganoderic acid G. The compound structural formula of Ganoderic acid delta is , the chemical structure of Ganoderic acid H is , the chemical structure of Lucidone B is , the chemical structure of Ganoderic acid J is The chemical structure of Ganoderic acid G is The chemical structure of ganoderic acid C2 is ; The chemical structural formula of ganoderic acid A is ; The chemical structural formula of ganoderic acid B is .

[0039] 3. The Ganoderma lucidum samples of Examples 1 to 3 are standard samples of Ganoderma lucidum type, referred to as standard samples; 4. The relative content analysis data of triterpenoid components of each type of Ganoderma lucidum obtained using the Ganoderma lucidum samples (wild (YS), understory cultivation (LX), greenhouse facility cultivation (DP), indoor factory cultivation (SN)), markers and methods of Examples 1 to 3 (including original data, analysis comparison data and analysis graphs), the principal component analysis data results of wild Ganoderma lucidum, the principal component analysis data results of each type of Ganoderma lucidum, and the extract, triterpene, and polysaccharide content data and analysis data of each type of Ganoderma lucidum measured according to the method specified in the 2020 edition of the "Chinese Pharmacopoeia".

[0040] 5. Determination method of the content of markers of each type of Ganoderma lucidum The content determination method of Ganoderic acid delta, Ganoderic acid H, Lucidone B, Ganoderic acid J, Ganoderic acid G, Ganoderic acid C2, Ganoderic acid A, and Ganoderic acid B is determined by HPLC quantitative analysis. The high performance liquid chromatography (General Rule 0512) in the Chinese Pharmacopoeia is a commonly used drug analysis method. Of course, the determination method can also be referred to and carried out according to the literature record of Tianjin University of Traditional Chinese Medicine published in the Chinese Journal of Traditional Chinese Medicine in October 2024, entitled "Qualitative Analysis and HPLC Content Determination of Triterpenoids in Ganoderma Lucidum Spores by UPLC-Q-TOF-MS / MS".

[0041] The content of active ingredients (extract, triterpenoids, and polysaccharide content) is determined according to the method specified in the 2020 edition of the Chinese Pharmacopoeia.

[0042] Example 5: Analysis and identification of relative contents of triterpenoid components of Ganoderma lucidum from different sources 1. Ganoderma lucidum medicinal materials: Ganoderma samples from different sources (species identified as red Ganoderma) were collected, and each type of identification sample included 5 parallel samples; Ganoderma from different sources included wild Ganoderma, Ganoderma cultivated in simulated forest environment, Ganoderma cultivated in greenhouses, and Ganoderma cultivated in indoor factories. They were numbered WZ01-WZ20 before being sent for inspection, and the sample inspection was carried out in a blind delivery manner. As shown in Table 2.

[0043] Table 2 Ganoderma lucidum samples from different sources

[0044] 2. Ganoderma lucidum sample processing Ganoderma lucidum samples from different sources were treated simultaneously with the standard samples of Example 4, namely, wild (YS), forest-cultivated (LX), greenhouse-cultivated (DP), and indoor factory-cultivated (SN); the treatment method was the same as that of Example 1.

[0045] 3. Standard sample testing The method for obtaining the original data of the Ganoderma lucidum standard sample and the method for determining the relative content of the triterpenoid components of Ganoderma lucidum are consistent with those in Example 1.

[0046] 4. Analysis results of relative contents of triterpenoids in Ganoderma lucidum The total ion current (TIC) of each sample was used for normalization and analysis to obtain Figure 8 Comparison of the relative contents of triterpenoids in Ganoderma lucidum under positive ion mode Fig. 9 Comparison of relative contents of triterpenoid components in Ganoderma lucidum under negative ion mode.

[0047] Figure 8It can be seen that from the relative content analysis results of the ganoderic acid C2 marker, the relative content determination values ​​of samples WZ01-05 and WZ20 are consistent with those of YS (wild Ganoderma lucidum); From the analysis results of the relative content of the marker ganoderic acid A, the relative content of samples WZ01-05 and WZ20 was similar to that of YS (wild Ganoderma), and the relative content of samples WZ02 and WZ05 was much higher than that of YS (wild Ganoderma); According to the relative content analysis results of Ganoderic_acid_delta marker, the relative content determination values ​​of WZ01-05 samples and YS (wild Ganoderma lucidum) are similar, and the relative content determination value of WZ05 is much higher than that of YS (wild Ganoderma lucidum); In terms of the relative content analysis results of the Ganoderic_acid H marker, the relative content determination values ​​of the WZ01-05 sample and YS (wild Ganoderma lucidum) were similar, both lower than those of other Ganoderma lucidum types; In terms of the relative content analysis results of the Lucidone_B marker, the relative content determination values ​​of samples WZ01-05, WZ13, and WZ18 were similar to those of YS (wild Ganoderma lucidum), which were all lower than those of other Ganoderma lucidum types; According to the relative content analysis results of Ganoderic_acid_J marker, the relative content values ​​of WZ01-05 and WZ20 samples were similar to those of YS (wild Ganoderma lucidum), and the relative content values ​​of WZ02, WZ04, and WZ05 were much higher than those of YS (wild Ganoderma lucidum); From the results of marker analysis in the positive ion mode, Ganoderic_acid_delta and Ganoderic_acid H alone can identify wild Ganoderma lucidum. Ganoderic acid C2, Ganoderic acid A, Lucidone_B, and Ganoderic_acid_J alone can also identify wild Ganoderma lucidum, but the accuracy is slightly lower than that of Ganoderic_acid_delta and Ganoderic_acid H. However, if any one or a combination of Ganoderic_acid_delta and Ganoderic_acid H is labeled with any one or a combination of several other markers, the accuracy of the labeling can be greatly improved.

[0048] Fig. 9 It can be seen that from the relative content analysis results of the ganoderic acid B marker, the relative content determination values ​​of the WZ01-05 sample and the YS (wild Ganoderma lucidum) are consistent; According to the relative content analysis results of the ganoderic acid C2 marker, the relative content determination values ​​of the WZ01-05 sample and the YS (wild ganoderma lucidum) were consistent; According to the relative content analysis results of the marker ganoderic acid G, the relative content of WZ01-08 sample was consistent with that of YS (wild ganoderma); From the analysis results of markers in negative ion mode, wild Ganoderma lucidum can be identified by labeling with ganoderic acid B and ganoderic acid C2 alone, and wild Ganoderma lucidum can also be identified by labeling with ganoderic acid G marker alone, but the accuracy is slightly lower than that of ganoderic acid B and ganoderic acid C2. However, if any one or a combination of ganoderic acid B and ganoderic acid C2 is combined with other ganoderic acid G markers, the accuracy of labeling can be greatly improved. Of course, it is also possible to combine the markers in positive ion mode with the markers in negative ion mode to greatly improve the accuracy of labeling.

[0049] 5. Content of Active Ingredients in Ganoderma Lucidum The contents of extracts, triterpenes and polysaccharides in Ganoderma lucidum and other cultivated varieties were determined according to the methods specified in the 2020 edition of the Chinese Pharmacopoeia.

[0050] Obtain a comparison chart of the active ingredient content of each type of Ganoderma lucidum, such as Fig.10 As shown: the extract content of WZ01-05 is basically consistent with that of wild Ganoderma lucidum, and has obvious differences from other types of Ganoderma lucidum extracts. Wild Ganoderma lucidum can be identified by using the extract alone as a marker; the triterpenoid components and total polysaccharides are also different, which are consistent with the content of wild Ganoderma lucidum. WZ01-05 can be identified as a wild Ganoderma lucidum type, but the difference is not obvious.

[0051] Example 6: Analysis and identification of effective components of Ganoderma lucidum from different sources 1. The method of obtaining the Ganoderma lucidum medicinal materials, sample processing, and raw data is consistent with that in Example 5.

[0052] 2. Data Processing and Analysis The raw data were converted into mzXML format using ProteoWizard software and processed by the XCMS program developed based on R language for peak detection, extraction, calibration and integration. The self-built BiotreeDB MS2 database was then applied for metabolite annotation, and the cutoff value of annotation was set to 0.3. The chemical components in the Ganoderma lucidum sample extracts were identified using the self-built BiotreeDB MS2 database, HMDB, Massbank and Chemspider databases.

[0053] The total ion current (TIC) of each sample was used for normalization. After obtaining the sorted data, the data were converted into logarithmic (log) and centered (CTR) formats, and then the sample data were subjected to principal component analysis (PCA) together with the database using SIMCA software (V15.0.2, Sartorius Stedim Data Analytics AB, Umea, Sweden). Fig.11 The principal component analysis score of Ganoderma lucidum in positive ion mode and Fig.12 The principal component analysis score of Ganoderma lucidum in negative ion mode. In order to distinguish the sample numbers more clearly at the clustering points of the samples, the enlarged image is placed in the rectangular frame, and the arrow points from the rectangular frame to be enlarged to the enlarged image.

[0054] 3. Results of principal component analysis It can be seen that the QC samples are clustered together, indicating that the instrument is running stably.

[0055] from Fig.11 , Fig.12 It can be seen that WZ01-05 is clustered with samples of wild Ganoderma lucidum, WZ06-10 is clustered with samples cultivated in greenhouse facilities, WZ11-15 is clustered with Ganoderma lucidum cultivated in simulated environment under forest, and WZ15-20 is clustered with Ganoderma lucidum cultivated in indoor factory. The distinction is obvious and there is no overlap. It can be seen that principal component analysis can further distinguish and identify various types of Ganoderma lucidum. Combining principal component analysis with relative content analysis of markers for joint identification can greatly improve the accuracy of identification.

[0056] Of course, HPLC quantitative analysis can also be used to determine the content of Ganoderic acid delta, Ganoderic acid H, Lucidone B, Ganoderic acid J, Ganoderic acid G, Ganoderic acid C2, Ganoderic acid A, and Ganoderic acid B in each type of Ganoderma lucidum, and then compare them with the marker content of each type of Ganoderma lucidum standard sample in the database to identify the type of Ganoderma lucidum.

[0057] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention. These should also be regarded as the scope of protection of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A marker for identifying the type of Ganoderma lucidum, characterized in that: The invention relates to any one compound of Ganoderic acid delta and Ganoderic acid H or a combination of the two compounds.

2. The Ganoderma lucidum type identification marker according to claim 1, characterized in that: It also includes any one substance or a combination of any several substances among Lucidone B, Ganoderic acid J, Ganoderic acid G, Ganoderic acid C2, Ganoderic acid A, Ganoderic acid B, and extracts.

3. A method for identifying the type of Ganoderma lucidum using the Ganoderma lucidum type identification marker according to any one of claims 1 or 2.

4. The method for identifying the types of Ganoderma lucidum according to claim 3, characterized in that: The contents of Ganoderic aciddelta, Ganoderic acid H, Lucidone B, Ganoderic acid J, Ganoderic acid G, Ganoderic acid C2, Ganoderic acid A and Ganoderic acid B are all detected by UHPLC-Q / TOF-MS mass spectrometry technology.

5. The method for identifying the types of Ganoderma lucidum according to claim 4, characterized in that: The relative content analysis diagram was obtained by normalizing the total ion current.

6. The method for identifying the types of Ganoderma lucidum according to claim 5, characterized in that: A principal component analysis step is also included.

7. A Ganoderma lucidum identification database constructed according to any one of the Ganoderma lucidum type identification markers according to claim 1 or 2.

8. The Ganoderma lucidum identification database according to claim 7, characterized in that: It also includes the method for identifying the type of Ganoderma lucidum according to claim 4, 5 or 6.

9. The Ganoderma lucidum identification database according to claim 8, characterized in that: It also includes the content values ​​of any one or several substances in the extracts of various types of Ganoderma lucidum, including Ganoderic acid delta, Ganoderic acid H, Lucidone B, Ganoderic acid J, Ganoderic acid G, Ganoderic acid C2, Ganoderic acid A, Ganoderic acid B, and other substances.

10. The Ganoderma lucidum type identification marker according to claim 1, characterized in that: The compound structural formula of Ganoderic aciddelta is , the chemical structural formula of the Ganoderic acid H is , the chemical structural formula of Lucidone B is , the chemical structural formula of the Ganoderic acid J is , the chemical structural formula of the Ganoderic acid G is .

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