A method for simultaneous detection of seven compounds in Hericium erinaceus

Abstract

The present invention provides three compounds extracted from Hericium erinaceus, namely erinacerin X, erinacerin Y, and erinacerin Z, and also provides a method for simultaneously detecting seven compounds in Hericium erinaceus, the method comprising the following steps: (1) a pretreatment step of the Hericium erinaceus sample; (2) an ultra-high performance liquid chromatography detection step; (3) a compound mass spectrometry information acquisition step; (4) an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry analysis method establishment step; and (5) a data analysis step. The method provided by the present invention can simultaneously perform accurate qualitative and quantitative analysis on the seven compounds produced by metabolism in the mycelium and fruiting body of Hericium erinaceus, with high sensitivity and strong specificity, thereby providing a rapid and accurate detection and analysis method for basic research on Hericium erinaceus and related product development.

Description

Technical Field

[0001] The present invention relates to the field of detection and analysis of effective components of edible and medicinal fungi, and in particular to a method for simultaneously detecting seven compounds in Hericium erinaceus. Background Art

[0002] Hericium erinaceus (Bull.) Pers., a fungus of the genus Hericium, is a traditional edible and medicinal fungus in my country. The Compendium of Materia Medica records that Hericium erinaceus has the benefits of "benefiting the five internal organs and aiding digestion." With the continuous advancement of science and technology, more nutritional, medicinal, and health benefits of Hericium erinaceus have been verified and explored. Previously, a variety of small molecule compounds, such as alkaloids, terpenes, and pyrones, have been discovered from the mycelium and fruiting bodies of Hericium erinaceus. These compounds have various pharmacological activities. For example, the compounds isohericerin (formerly known as hericerin) and N-de phenylethylisohericerin have both been shown to exhibit α-glucosidase inhibitory activity. Isohericerin and corallocin A can enhance the production of nerve growth factor (NGF) and promote neurite outgrowth in N2a neurons.

[0003] These compounds in Hericium erinaceus will form the basis for the future development of blood sugar-lowering and anti-aging drugs, health foods, or functional foods. Therefore, it is necessary to establish a precise quantitative method for the simultaneous detection of these alkaloid and pyranoid active compounds to provide a quality control standard for the development of Hericium erinaceus fruiting bodies or mycelium raw materials and future related products. Summary of the Invention

[0004] The present invention first provides three compounds, erinacerin X, erinacerin Y, and erinacerin Z, which are Compound 1, Compound 5, and Compound 6, respectively:

[0005]

[0006]

[0007] The three compounds provided by the present invention are extracted and prepared from Hericium erinaceus mycelium. The specific preparation method comprises the following steps:

[0008] (1) Extraction and extraction of Hericium erinaceus mycelium

[0009] The dried Hericium erinaceus mycelia were soaked and extracted with 95% by volume ethanol aqueous solution at room temperature for 3 times, each time for 24 hours, and the extracts were combined and concentrated to obtain Hericium erinaceus mycelia ethanol extract.

[0010] The Hericium erinaceus alcohol extract was redissolved in a 75% by volume ethanol aqueous solution to prepare a suspension, extracted three times with petroleum ether, the petroleum ether solutions were combined, and concentrated under reduced pressure to obtain a petroleum ether extract; the remaining part was extracted three times with ethyl acetate, the ethyl acetate solutions were combined, and concentrated under reduced pressure to obtain an ethyl acetate extract.

[0011] (2) Preparation of 7 compounds

[0012] The ethyl acetate extract was separated by medium pressure chromatography using YMC C18 (50 μm, 12 nm) as a filler and a water (A)-acetonitrile (B) gradient elution (0 min: 80% A, 20% B; 30 min: 75% A, 25% B; 36 min: 60% A, 40% B; 48 min: 55% A, 45% B; 93 min: 10% A, 90% B; 98 min: 0% A, 100% B) at a flow rate of 200 ml / min. Fractions were collected every 400 ml, for a total of 55 fractions, which were numbered in the order received and analyzed by thin layer chromatography. Similar fractions were combined to obtain Fr1-Fr15.

[0013] 15 components: Fr.1 (1-6, 21.46g), Fr.2 (7-12, 1.21g), Fr.3 (13-14, 0.22g), Fr.4 (15-19, 0 .34g), Fr.5(20-22, 0.33g), Fr.6(23-24, 0.58g), Fr.7(25-27, 0.76g), Fr.8(28-29 , 0.27g), Fr.9 (30-33, 0.48g), Fr.10 (34-37, 0.63g), Fr.11 (38-40, 0.72g), Fr.12 ( 41-44, 1.43g), Fr.13 (45-47, 0.56g), Fr.14 (48-49, 0.25g), Fr.15 (50-55, 0.22g).

[0014] Isolation and purification of Fr.7

[0015] Fr.7 was separated by reverse phase chromatography using a ZORBAX Eclipse Plus C18 (5μm, 21.2mm×250mm) column, an elution system of 0.01% glacial acetic acid water (A) and acetonitrile (B), an elution program of 0 min: 80% A, 20% B; 60 min: 70% A, 30% B, an elution flow rate of 15 mL / min, a detection wavelength of 210 nm, and the chromatographic peak with a retention time (RT) of approximately 41.853 min was collected to obtain Fr.7-1.

[0016] Fr.7-1 was separated by reverse-phase chromatography on a ZORBAX Eclipse Plus C18 column (5 μm, 21.2 mm × 250 mm). The elution system consisted of 0.01% glacial acetic acid in water (A) and acetonitrile (B). The elution procedure was isocratic, with an elution ratio of 79% A and 21% B at a flow rate of 15 mL / min and detection at a wavelength of 210 nm. The peak at approximately RT 61.306 min was collected to yield compound 1 (4.8 mg).

[0017] Isolation and purification of Fr.10

[0018] Fr.10 was separated by reverse phase chromatography using a ZORBAX Eclipse Plus C18 (5μm, 21.2mm×250mm) column, an elution system of 0.01% glacial acetic acid water (A) and acetonitrile (B), and an elution program of 0 min: 63% A, 37% B; 40 min: 58% A, 42% B; 45 min: 58% A, 42% B. The elution flow rate was 15 mL / min, the detection wavelength was 220 nm, and the chromatographic peak with a RT of about 33.674 min was collected to obtain Fr.10-1.

[0019] Fr.10-1 was separated by reverse phase chromatography using a ZORBAX Eclipse Plus C18 column (5 μm, 21.2 mm × 250 mm) and an elution system of 0.01% glacial acetic acid water (A) and acetonitrile (B). The elution procedure was 0 min: 63% A, 37% B; 40 min: 60% A, 40% B. The elution flow rate was 15 mL / min, the detection wavelength was 220 nm, and the chromatographic peak with an RT of approximately 37.096 min was collected to obtain compound 2 (13.8 mg).

[0020] Isolation and purification of Fr.11

[0021] Fr.11 was separated by reverse phase chromatography using a ZORBAX Eclipse Plus C18 (5μm, 21.2mm×250mm) column, an elution system of 0.01% glacial acetic acid water (A) and acetonitrile (B), an elution program of 0min: 58% A, 42% B; 40min: 51% A, 49% B, an elution flow rate of 15mL / min, a detection wavelength of 220nm, and the chromatographic peak with a RT of about 37.199min was collected to obtain Fr.11-1.

[0022] Fr.11-1 was separated by gel chromatography with methanol as the elution solvent. After TLC detection, the same fractions were combined to obtain compound 3 (17.5 mg).

[0023] Isolation and purification of Fr.12

[0024] Fr.12 was separated by reverse phase chromatography using a ZORBAX Eclipse Plus C18 column (5 μm, 21.2 mm × 250 mm), an elution system of 0.01% glacial acetic acid water (A) and acetonitrile (B), an elution program of 0 min: 55% A, 45% B; 50 min: 38% A, 62% B, an elution flow rate of 20 mL / min, a detection wavelength of 201 nm, and a chromatographic peak at around 22.346 min was collected to obtain compound 4 (26.8 mg). A chromatographic peak at around 40.997 min of RT was collected to obtain compound 5 (14.1 mg). A chromatographic peak at around 45.321 min of RT was collected to obtain compound 6 (8.9 mg).

[0025] Isolation and purification of Fr.15

[0026] Fr.15 was recrystallized from ethanol to give compound 7 (33.0 mg).

[0027] The structural formulas of the above compounds 1-7 are shown in the attached Figure 1 Among them, compound 1, compound 5 and compound 6 are new compounds discovered for the first time in the present invention. Compounds 1, 5 and 6 have significant hypoglycemic effects and can be used to prepare drugs for preventing or treating diabetes, or to prepare health foods for controlling blood sugar.

[0028] The present invention also aims to provide a method for simultaneously detecting seven compounds in Hericium erinaceus, the method comprising the following steps:

[0029] (1) Pretreatment steps of Hericium erinaceus samples: add Hericium erinaceus fruiting bodies or mycelium to ethanol for ultrasonic extraction or heating extraction at 60°C, take the supernatant, filter, and the obtained filtrate is the sample solution to be tested; prepare a mixed reference solution of 7 compounds;

[0030] (2) Ultra-high performance liquid chromatography detection step: The sample solution and the mixed reference solution of the seven compounds were separated by ultra-high performance liquid chromatography to obtain a good separation degree;

[0031] (3) Compound mass spectrometry information acquisition step: Using the mass spectrometry optimization software Agilent Optimizer, the seven compounds were scanned for parent ions, detected for daughter ion pairs, and searched for the optimal collision energy;

[0032] (4) Steps for establishing the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry analytical method: In the data acquisition software Agilent MassHunter Data Acquisition, under ultra-high performance liquid chromatography analysis conditions and triple quadrupole mass spectrometry detection conditions, retention time, parent ion, daughter ion pair, and collision energy data were imported to establish a dynamic multiple reaction monitoring (DMRM) monitoring and analytical determination method for the seven compounds; at the same time, mixed reference solutions and test sample solutions of the seven compounds were loaded for determination;

[0033] (5) Data analysis step: using the quantitative analysis software Agilent MassHunter Quantitative Analysis, standard curves of seven compounds were created to validate the established method and quantitatively analyze the sample solutions;

[0034] The seven compounds described are:

[0035] Erinacerin X(C 14 H 16 O6, compound 1);

[0036] Corallocin A(C 19 H 22 O6 compound 2);

[0037] Hericerin A(C 21 H 29 NO4, compound 3);

[0038] N-de phenylethyl isohericerin(C 19 H 25 NO3, compound 4);

[0039] Erinacerin Y(C 25 H 35 NO5, compound 5);

[0040] Erinacerin Z(C 24 H 33 NO5, compound 6);

[0041] Isohericerin (C 27 H 33 NO3, compound 7);

[0042] Preferably, the pretreatment step of the Hericium erinaceus sample in step (1) is to vacuum freeze-dry the mycelium or fruiting body sample to be tested, add ethanol at a solid-liquid ratio of 1:20-40 (weight g: volume ml) (preferably 1:20), ultrasonically extract for 10-90 minutes or heat extract at 60°C for 10-90 minutes (preferably: ultrasonic for 60 minutes), and filter the extract with an organic phase microporous filter membrane with a pore size of 0.22 μm to obtain a sample solution to be tested;

[0043] Preferably, the preparation method of the mixed reference solution of the seven compounds in step (1) is as follows: accurately weigh the seven compounds respectively, prepare a mixed standard solution with a concentration of 5 μg / mL using mass spectrometry grade methanol (wherein the concentration of each compound is 5 μg / mL), and then dilute stepwise to obtain mixed standard working solutions with concentrations of 2 μg / mL, 1 μg / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, and 20 ng / mL for the seven compounds, respectively;

[0044] Preferably, the chromatographic conditions for ultra-high performance liquid chromatography separation in step (2) are as follows: an Agilent ZORBAX Eclipse Plus C18 column, 1.8 μm, 2.1×150 mm, detection wavelength: 240 nm; column temperature: 35° C.; sample load: 2 μL; flow rate: 0.4 mL / min; mobile phase A: 0.01% glacial acetic acid; mobile phase B: acetonitrile; elution program: 0 min: 80% A, 20% B; 12 min: 75% A, 25% B; 14 min: 63% A, 37% B; 19 min: 55% A, 45% B; 38 min: 10% A, 90% B; 40 min: 0% A, 100% B;

[0045] Preferably, the retention time, parent ion, daughter ion pair and collision energy information of each compound required for analysis in step (3) are obtained by mass spectrometry optimization software (Agilent Optimizer) as follows:

[0046] Erinacerin X(C 14 H 16 O6, compound 1): retention time: 11.65 min; parent ion: 279.1; quantitative ion: 245.0, collision energy: 17; qualifier ion: 173.0, collision energy 21;

[0047] Corallocin A(C 19 H 22 O6 compound 2): retention time: 19.98 min; parent ion: 345.1; quantitative ion: 300.9, collision energy: 13; qualifier ion: 121.0, collision energy 29;

[0048] Hericerin A(C 21 H 29 NO4, compound 3): retention time: 23.27 min; parent ion: 358.2; quantitative ion: 274.0, collision energy: 29; qualifier ion: 219.8, collision energy 33;

[0049] N-de phenylethyl isohericerin(C 19 H 25 NO3, compound 4): retention time: 23.89 min; parent ion: 314.2; quantitative ion: 229.9, collision energy: 29; qualifier ion: 175.9, collision energy 25;

[0050] Erinacerin Y(C 25 H 35 NO5, compound 5): retention time: 28.21 min; parent ion: 428.2; quantitative ion: 343.9, collision energy: 37; qualifier ion: 297.9, collision energy 37;

[0051] Erinacerin Z(C 24 H 33 NO5, compound 6): retention time: 28.87 min; parent ion: 414.2; quantitative ion: 340.1, collision energy: 25; qualifier ion: 256.0, collision energy 41;

[0052] Isohericerin (C 27 H 33 NO3, compound 7); retention time: 30.41 min; parent ion: 418.2; quantitative ion: 334.0, collision energy: 19; qualifier ion: 280.0, collision energy 33;

[0053] Preferably, in step (4), ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used as the analytical detection instrument, wherein the chromatographic conditions of ultra-high performance liquid chromatography separation (the same as the chromatographic conditions in step (2)) are: Agilent ZORBAX Eclipse Plus C18 chromatographic column, 1.8 μm, 2.1×150 mm, detection wavelength: 240 nm; column temperature: 35° C.; sample load: 2 μL; flow rate: 0.4 mL / min; mobile phase A: 0.01% glacial acetic acid; mobile phase B: acetonitrile; elution program: 0 min: 80% A, 20% B; 12 min: 75% A, 25% B; 14 min: 63% A, 37% B; 19 min: 55% A, 45% B; 38 min: 10% A, 90% B; 40 min: 0% A, 100% B; wherein the mass spectrometry conditions are: electrospray ionization source (AJS ESI) was used as the ion source, and the detection was carried out in negative ion mode. The dynamic multiple reaction monitoring (DMRM) mode was selected, with the capillary voltage and outlet voltage being 3500 V and 380 V, respectively, the nozzle voltage being 2000 V, the dryer temperature and flow rate being 200 °C and 16 L / min, respectively, and the sheath gas temperature and flow rate being 350 °C and 12 L / min, respectively.

[0054] The identification basis of a method provided by the present invention for simultaneously detecting 7 compounds in Hericium erinaceus is: using ultra-performance liquid chromatography-triple quadrupole mass spectrometry to detect, on the basis of qualitative identification of compounds by retention time, further qualitative and quantitative analysis is performed based on the molecular ion information of the compounds, and precise targeted detection of compounds.

[0055] Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-TQMS) enables accurate qualitative and quantitative detection of compounds not only through retention time but also through mass spectral information such as parent and daughter ions. Therefore, the present invention uses UPLC-TQMS to establish a method for the simultaneous detection of seven compounds in the mycelium or fruiting bodies of Hericium erinaceus. This enables quantitative analysis in biomaterials with high levels of impurities and similar structures of target compounds.

[0056] The beneficial effects of the present invention are as follows: the technical solution of the present invention combines the high separation ability of liquid chromatography and the function of mass spectrometry to provide structural information, which can obtain rich and effective compound structural information, has the advantages of high sensitivity and strong selectivity, and can effectively overcome the shortcomings of chemical methods or liquid chromatography methods, thereby providing accurate and reliable detection and analysis methods for Hericium erinaceus related research and products. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 、Structural formulas of 7 compounds of Hericium erinaceus

[0058] Figure 2 DMRM acquisition diagram of 7 compounds of Hericium erinaceus

[0059] Among them, compound 1: Erinacerin X;

[0060] Compound 2: Corallocin A;

[0061] Compound 3: Hericerin A;

[0062] Compound 4: N-de phenylethyl isohericerin;

[0063] Compound 5: Erincerin Y;

[0064] Compound 6: Erinacerin Z;

[0065] Compound 7: Isohericerin; DETAILED DESCRIPTION

[0066] The following is a detailed description of the technical solution of the present invention. However, the following does not limit the technical solution of the present invention and the scope of protection of the present invention cannot be limited by them. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0068] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0069] Potato agar (PDA) with glucose: purchased from Beijing Solebow Technology Co., Ltd.

[0070] Glucose, soluble starch, yeast, MgSO4·H2O, KH2PO4, CaSO4, and analytical grade ethanol, chloroform, petroleum ether, acetone, ethanol, ethanol, acetonitrile, and glacial acetic acid were purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0071] α-Glucosidase (33.7 U / mg) and acarbose were purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0072] Phosphate buffered saline (PBS) and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Company, USA.

[0073] 4-Nitrophenyl-α-D-pyranoglucopyranoside (PNPG): purchased from Tokyo Chemical Industry Development Co., Ltd. (Shanghai)

[0074] Chromatographic grade methanol and acetonitrile were purchased from Shanghai Anpu Laboratory Technology Co., Ltd.

[0075] Deuterated chloroform: purchased from Sigma;

[0076] YMC*GEL C18 silica gel (50 μm, 12 nm): YMC Co., Ltd., Japan;

[0077] Sephadex LH-20 gel (70 μm): GE, USA;

[0078] Zorbax Eclipse Plus C18 analytical column (1.8 μm, 2.1 × 150 mm): Agilent, USA;

[0079] Zorbax Eclipse Plus-C18 semi-preparative column (5 μm, 21.2 mm × 250 mm): Agilent, USA;

[0080] C-605-620-660 medium pressure preparative chromatography: Switzerland company;

[0081] LC3000 medium and high pressure preparative chromatograph: Beijing Chuangxin Tongheng Technology Co., Ltd.

[0082] Agilent LC1290 infinity II ultra-high performance liquid chromatograph: Agilent, USA;

[0083] Agilent 6495 triple quadrupole mass spectrometer: Agilent, USA;

[0084] Bruker AV II-600 NMR spectrometer: Bruker, Germany;

[0085] Synergy HT multifunctional microplate reader: BIO-TEK, USA;

[0086] Thermo Scientific Q Exactive HF Orbitrap-FTMS high-resolution mass spectrometer: Thermo Scientific, USA

[0087] Source of Hericium erinaceus mycelium: strain 1: Hericium erinaceus, strain number: 7080, the strain is currently preserved in the National Edible Fungi Germplasm Resource Bank (Shanghai).

[0088] Example 1 Hericium erinaceus mycelium fermentation

[0089] Culture medium formula: soluble starch 3%, glucose 1.2%, yeast 3%, MgSO4·7H2O 0.20%, KH2PO4 0.20% (all percentages are by weight), balance water, natural pH.

[0090] The strain (strain 1) stored on PDA medium in a refrigerator at 4°C was activated and cultured in a shake flask under first-level shaking conditions for 7 days (150 r / min, 26°C) in the dark. It was transferred to a second-level shake flask at a 10% inoculum and cultured for 7 days (150 r / min, 26°C), and then switched to static culture for 21 days to obtain mycelium.

[0091] Example 2 Preparation of 7 compounds

[0092] (1) Extraction and extraction of Hericium erinaceus mycelium

[0093] The dried mycelium (strain 1) obtained by fermentation in Example 1 was soaked and extracted three times with a 95% ethanol aqueous solution at room temperature, each time for 24 hours. The extracts were combined and concentrated to obtain an alcohol extract of Hericium erinaceus mycelium.

[0094] The Hericium erinaceus alcohol extract was redissolved in 75% ethanol aqueous solution to prepare a suspension, extracted three times with petroleum ether, the petroleum ether solutions were combined, and concentrated under reduced pressure to obtain a petroleum ether extract; the remaining part was extracted three times with ethyl acetate, the ethyl acetate solutions were combined, and concentrated under reduced pressure to obtain an ethyl acetate extract.

[0095] (2) Preparation of 7 compounds

[0096] The ethyl acetate extract was separated by medium pressure chromatography using YMC C18 (50 μm, 12 nm) as a filler and a water (A)-acetonitrile (B) gradient elution (0 min: 80% A, 20% B; 30 min: 75% A, 25% B; 36 min:

[0097] The flow rate was 200 ml / min, and 400 mL fractions were collected. A total of 55 fractions were collected, numbered in the order received, and analyzed by thin-layer chromatography. Similar fractions were combined to obtain Fr1-Fr15.

[0098] 15 components: Fr.1 (1-6, 21.46g), Fr.2 (7-12, 1.21g), Fr.3 (13-14, 0.22g), Fr.4 (15-19, 0 .34g), Fr.5(20-22, 0.33g), Fr.6(23-24, 0.58g), Fr.7(25-27, 0.76g), Fr.8(28-29 , 0.27g), Fr.9 (30-33, 0.48g), Fr.10 (34-37, 0.63g), Fr.11 (38-40, 0.72g), Fr.12 ( 41-44, 1.43g), Fr.13 (45-47, 0.56g), Fr.14 (48-49, 0.25g), Fr.15 (50-55, 0.22g).

[0099] Isolation and purification of Fr.7

[0100] Fr.7 was separated by reverse phase chromatography using a ZORBAX Eclipse Plus C18 (5μm, 21.2mm×250mm) column, an elution system of 0.01% glacial acetic acid water (A) and acetonitrile (B), an elution program of 0 min: 80% A, 20% B; 60 min: 70% A, 30% B, an elution flow rate of 15 mL / min, a detection wavelength of 210 nm, and the chromatographic peak with a retention time (RT) of approximately 41.853 min was collected to obtain Fr.7-1.

[0101] Fr.7-1 was separated by reverse-phase chromatography on a ZORBAX Eclipse Plus C18 column (5 μm, 21.2 mm × 250 mm). The elution system consisted of 0.01% glacial acetic acid in water (A) and acetonitrile (B). The elution procedure was isocratic, with an elution ratio of 79% A and 21% B at a flow rate of 15 mL / min and detection at a wavelength of 210 nm. The peak at approximately RT 61.306 min was collected to yield compound 1 (4.8 mg).

[0102] Isolation and purification of Fr.10

[0103] Fr.10 was separated by reverse phase chromatography using a ZORBAX Eclipse Plus C18 (5μm, 21.2mm×250mm) column, an elution system of 0.01% glacial acetic acid water (A) and acetonitrile (B), and an elution program of 0 min: 63% A, 37% B; 40 min: 58% A, 42% B; 45 min: 58% A, 42% B. The elution flow rate was 15 mL / min, the detection wavelength was 220 nm, and the chromatographic peak with a RT of about 33.674 min was collected to obtain Fr.10-1.

[0104] Fr.10-1 was separated by reverse phase chromatography using a ZORBAX Eclipse Plus C18 column (5 μm, 21.2 mm × 250 mm) and an elution system of 0.01% glacial acetic acid water (A) and acetonitrile (B). The elution procedure was 0 min: 63% A, 37% B; 40 min: 60% A, 40% B. The elution flow rate was 15 mL / min, the detection wavelength was 220 nm, and the chromatographic peak with an RT of approximately 37.096 min was collected to obtain compound 2 (13.8 mg).

[0105] Isolation and purification of Fr.11

[0106] Fr.11 was separated by reverse phase chromatography using a ZORBAX Eclipse Plus C18 (5μm, 21.2mm×250mm) column, an elution system of 0.01% glacial acetic acid water (A) and acetonitrile (B), an elution program of 0min: 58% A, 42% B; 40min: 51% A, 49% B, an elution flow rate of 15mL / min, a detection wavelength of 220nm, and the chromatographic peak with a RT of about 37.199min was collected to obtain Fr.11-1.

[0107] Fr.11-1 was separated by gel chromatography with methanol as the elution solvent. After TLC detection, the same fractions were combined to obtain compound 3 (17.5 mg).

[0108] Isolation and purification of Fr.12

[0109] Fr.12 was separated by reverse phase chromatography using a ZORBAX Eclipse Plus C18 column (5 μm, 21.2 mm × 250 mm), an elution system of 0.01% glacial acetic acid water (A) and acetonitrile (B), an elution program of 0 min: 55% A, 45% B; 50 min: 38% A, 62% B, an elution flow rate of 20 mL / min, a detection wavelength of 201 nm, and a chromatographic peak at around 22.346 min was collected to obtain compound 4 (26.8 mg). A chromatographic peak at around 40.997 min of RT was collected to obtain compound 5 (14.1 mg). A chromatographic peak at around 45.321 min of RT was collected to obtain compound 6 (8.9 mg).

[0110] Isolation and purification of Fr.15

[0111] Fr.15 was recrystallized from ethanol to give compound 7 (33.0 mg).

[0112] (3) Confirmation of compounds 1-7.

[0113] Using mass spectrometry and nuclear magnetic resonance (NMR) detection, we analyzed the structures of seven compounds obtained from Hericium erinaceus mycelium and determined their precise chemical structures. The specific confirmation information of the seven compounds is as follows:

[0114] Compound 1erinacerin X

[0115] The molecular formula is C 14 H 16 O6, white amorphous powder, easily soluble in chloroform and methanol. HRESIMS m / z: 279.0876 [MH] - (C 14 H 15 O6, calculated value 279.0874). 13 C-NMR(CDCl3,125MHz)δ:182.3(C-4'),172.3(C-1),159.6(C-6),150.2(C-4),127.7(C-3a),125.1(C-7a) ,122.2(C-5),98.1(C-7),68.5(C-3),56.2(C-OCH3),38.5(C-3'),32.0(C-2'),21.3(C-1'),17.6(C-5'); 1 H-NMR(CDCl3,500MHz)δ:6.94(1H,s,H-7),5.25(1H,s,H-3),3.86(3H,s,H-OCH3),2.80(1H,m, H-1'), 2.69 (1H, m, H-1'), 2.56 (1H, m, H-3'), 1.78 (1H, m, H-2'), 1.26d (1H, d, J = 10.0Hz, H-5').

[0116] Compound 2corallocin A

[0117] The molecular formula is C 19 H 22 O6, light yellow oil, easily soluble in methanol. ESI positive ion mode m / z: 369.0

[0118] [M+Na] + . 13C-NMR(CDCl3,125MHz)δ:172.9C-8'),172.4(C-1),159.3(C-6),149.9(C-4),144.39(C-6'),137.09(C-3'),127.5(C-7'),127.3(C-3a),124.8 (C-7a),122.4(C-5),122.3(C-2'),98.6(C-7),68.4(C-3),56.2(C-OCH 3),38.1(C-4'),26.89(C-5'),22.9(C-1'),16.1(C-9'),12.3(C-10'); 1 H-NMR(CDCl3,500MHz)δ:6.92(1H,s,H-7),6.77(1H,t,J=5.0Hz,H-6'),5.22(1H,m,H-2'),5.21(1H,s,H-3),3.85(3H,s,H- OCH3),3.47(1H,d,J=5.0Hz,H-1'),2.33(1H,m,H-5'),2.19(1H,t,J=5.0Hz,H-4'),1.80(1H,s,H-9'),1.78(1H,s,H-10').

[0119] Compound 3hericerin A

[0120] The molecular formula is C 21 H 29 NO4, light yellow amorphous powder, easily soluble in chloroform and methanol. ESI positive ion mode m / z: 360.2 [M+H] + . 13 C-NMR(CDCl3,125MHz)δ:170.5(C-1),158.7(C-6),150.4(C-4),138.7(C-3' ),132.1(C-7'),131.5(C-3a),123.9(C-6'),121.5(C-7a),121.4(C-2'),119 .6(C-5),97.5(C-7),61.4(C-2”),56.1(C-OCH3),49.4(C-3),46.2(C-1”),39 .9(C-4'),26.5(C-5'),25.8(C-8'),22.9(C-1'),17.8(C-10'),16.3(C-9'); 1H-NMR (CDCl3, 500MHz) δ: 6.89 (1H, s, H-7), 5.22 (1H, t, J = 5.0 Hz, H-2'), 5.04 (1H, t, J =5.0Hz,H-6'),4.39(1H,s,H-3),3.86(1H,t,J=5.0Hz,H-2”),3.81(3H,s,H-OCH3),3. 71(1H,t,J=5.0Hz,H-1”),3.46(1H,d,J=5.0Hz,H-1’),2.09(1H,m,H-5’),2.05(1H,m ,H-5'),2.04(1H,m,H-4'),1.80(1H,s,H-9'),1.66(1H,s,H-8'),1.58(1H,s,H-10').

[0121] Compound 4N-de phenylethyl isohericerin

[0122] The molecular formula is C 19 H 25 NO3, colorless amorphous powder, easily soluble in chloroform and methanol. ESI positive ion mode m / z: 316.0 [M+H] + . 13 C-NMR (CDCl3, 125MHz) δ: 169.2 (C-1), 158.5 (C-6), 150.7 (C-4), 140.0 (C-3'), 132.4 (C-7'), 132.2 (C-3a), 123.7 (C-6'), 121.2 (C-7a), 121. 2(C-2'),118.4(C-5),97.8(C-7),56.3(C-OCH3),47.7(C-3),39.8(C- 4'),26.4(C-5'),25.9(C-8'),22.9(C-1'),17.9(C-10'),16.3(C-9'); 1 H-NMR(CDCl3,500MHz)δ:6.97(1H,s,H-7),5.25(1H,t,J=5.0Hz,H-2'),5.04(1H,t,J=5.0Hz,H-6'),4.34(1H,s,H-3),3.86(3H,s,H-OCH3),3. 50(1H,d,J=5.0Hz,H-1'),2.11(1H,m,H-5'),2.09(1H,m,H-4'),2.09( 1H,m,H-5'),1.82(1H,s,H-9'),1.67(1H,s,H-8'),1.59(1H,s,H-10').

[0123] Compound 5erinacerin Y

[0124] The molecular formula is C 25 H 35 NO5, light yellow oil, easily soluble in chloroform and methanol. HRESIMS m / z: 452.2404 [M+Na] + (C 25 H 35 NO5Na, calculated value 452.2407). 13 C-NMR(CDCl3,125MHz)δ:173.3(C-4”),169.2(C-1),158.5(C-6),150.7(C-4),140.0(C-3' ),132.4(C-7'),132.2(C-3a),123.7(C-6'),121.2(C-7a),121.2(C-2'),118.4(C-5),97. 8(C-7),60.7(C-5”),56.3(C-OCH3),47.7(C-3),42.0(C-1”),39.8(C-4’),31.6(C-3”),26 .4(C-5'),25.9(C-8'),23.9(C-2”),22.9(C-1’),17.9(C-10’),16.3(C-9’),14.3(C-6”); 1 H-NMR(CDCl3,500MHz)δ:6.97(1H,s,H-7),5.25(1H,t,J=12.0Hz,H-2'),5.03(1H,t,J=6.0Hz,H-6'),4.28(1H,s ,H-3),4.09(1H,dd,J=6.0,12.0Hz,H-5”),3.86(3H,s,H-OCH3),3.64(1H,t,J=12.0Hz,H-1”),3.49(1H,d,J=12. 0Hz,H-1'),2.37(1H,t,J=6.0Hz,H-3"),2.12(1H,m,H-5'),2.09(1H,t,J=6.0Hz,H-4'),2.09(1H,m,H-5'),2.00 (1H,t,J=12.0Hz,H-2”),1.81(1H,s,H-9’),1.66(1H,s,H-8’),1.58(1H,s,H-10’),1.23(1H,t,J=6.0Hz,H-6”).

[0125] Compound 6erinacerin Z

[0126] The molecular formula is C 24 H 33NO5, light yellow oil, easily soluble in chloroform and methanol. HRESIMS m / z: 438.2240 [M+Na] + (C 24 H 33 NO5Na, calculated value 438.2251). 13 C-NMR (CDCl3, 125MHz) δ:

[0127] 172.0(C-2”),169.2(C-1),158.5(C-6),150.6(C-4),140.0(C-3’),132.5(C-7’),131.7(C-3a),

[0128] 123.7(C-6'),121.9(C-7a),121.1(C-2'),118.6(C-5),97.9(C-7),61.5(C-3”),56.3(C-OCH3),49.5(C-1”),44. 5(C-3),39.8(C-4'),26.4(C-5'),25.9(C-8'),22.9(C-1'),17.9(C-10'),16.4(C-9'),16.0(C-1”),14.3(C-4”); 1 H-NMR(CDCl3,500MHz)δ:6.97(1H,s,H-7),5.25(1H,t,J=6.0Hz,H-2'),5.15(1H,m,H -1"),5.04(1H,t,J=6.0Hz,H-6'),4.44(1H,d,J=12.0Hz,H-3),4.30(1H,d,J=18.0Hz ,H-3),4.18(1H,dd,J=6.0,12.0Hz,H-3”),3.86(3H,s,H-OCH3),3.49(1H,d,J=6.0Hz ,H-1'),2.12(1H,m,H-5'),2.09(1H,m,H-4'),2.09(1H,m,H-5'),1.83(1H,s,H-9'),

[0129] 1.68(1H,s,H-8'), 1.60(1H,s,H-10'), 1.56(1H,d,J=12.0Hz,H-5”), 1.26(1H,t,J=6.0Hz,H-4”).

[0130] Compound 7isohericerin

[0131] The molecular formula is C 27 H 33NO3, colorless amorphous powder, easily soluble in chloroform and methanol. ESI positive ion mode m / z: 442.0 [M+Na] + . 13 C-NMR(CDCl3,125MHz)δ:169.0(C-1),158.5(C-6),150.7(C-4),139.7(C-3”),138.9(C-3’), 132.4(C-7'),132.3(C-3a),128.8(C-4”,8”),128.7(C-5”,7”),126.6(C-6”),123.8(C-6’), 121.3(C-2'),121.2(C-7a),118.4(C-5),97.7(C-7),56.2(C-OCH3),48.2(C-3),44.4(C-1”) ,39.8(C-4'),35.0(C-2”),26.4(C-5’),25.9(C-8’),22.9(C-1’),17.9(C-10’),16.3(C-9’); 1 H-NMR(CDCl3,500MHz)δ:7.27(1H,m,H-6”),7.26(1H,m,H-5”,7”),7.22(1H,m,H-4”,8”),6.96(1H,s ,H-7),5.24(1H,t,J=10.0Hz,H-2'),5.03(1H,t,J=5.0Hz,H-6'),4.15(1H,s,H-3),3.85(1H,t,J=10 .0Hz,H-1”),3.84(3H,s,H-OCH3),3.49(1H,d,J=10.0Hz,H-1’),2.97(1H,t,J=10.0Hz,H-2”),2.10( 1H,m,H-5'),2.08(1H,t,J=5.0Hz,H-4'),1.81(1H,s,H-9'),1.66(1H,s,H-8'),1.58(1H,s,H-10').

[0132] Example 3 Verification of the Hypoglycemic Activity of Compounds in Vitro

[0133] After accurately weighing each of the seven compounds, they were prepared into 2000 μM sample solutions with DMSO, and then diluted stepwise into 1000 μM, 500 μM, 200 μM, and 100 μM sample solutions.

[0134] Sample group: 20 μL of sample solution of varying concentrations was added to each 96-well plate. Then, 180 μL of PBS and 25 μL of α-glucosidase solution (0.2 U / mL) were added to each well. Mix thoroughly and incubate at room temperature for 10 minutes. Then, 25 μL of PNPG (23.2 mM in PBS) was added and the reaction was carried out at 37°C for 15 minutes. After completion of the reaction, the absorbance of each well was measured at 405 nm.

[0135] Positive control group: Replace the sample solution in the sample group with acarbose solution. Acarbose solution preparation method: Accurately weigh acarbose and dissolve it in DMSO to obtain a 10,000 μM acarbose solution. Then, dilute it with DMSO in a series of dilutions to 2,000 μM, 1,000 μM, 500 μM, 100 μM, 10 μM, 1 μM, and 0.5 μM acarbose solutions.

[0136] Blank group: 20 μL DMSO was used to replace the sample solution in the sample group.

[0137] Background groups included a blank background group, a sample background group, and an acarbose background group. All background groups replaced the α-glucosidase solution with 25 μL of PBS solution. For example, the blank background group replaced the α-glucosidase solution in the blank group with 25 μL of PBS solution.

[0138]

[0139] Experimental results:

[0140] Among the 7 compounds, compounds 2 (corallocins A), 4 (N-de phenylethyl isohericerin), 5 (erinacerin Y), 6 (erinacerin Z), and 7 (isohericerin) showed strong inhibitory activity against α-glucosidase. Among them, compound 7 had the strongest inhibitory ability, with an IC 50 The inhibitory ability of compounds 4, 6, 5, and 2 decreased in turn, and the IC 50 The values ​​were 31.29 μM, 31.73 μM, 40.08 μM, and 76.49 μM, respectively. Compounds 1 (erinacerin X) and 3 (hericerin A) showed poorer activity, with inhibition rates against α-glucosidase below 50% at the highest concentration tested (160 μM). Detailed results are shown in Table 9.

[0141] Example 4 Establishment of detection method

[0142] Preparation of mixed reference solution: Take the seven reference substances of the compound prepared in Example 2 and prepare a mixed standard solution with a concentration of 5 μg / mL using mass spectrometry grade methanol (wherein the concentration of each reference substance is 5 μg / mL), and dilute it stepwise with mass spectrometry grade methanol to mixed reference solutions with concentrations of 2 μg / mL, 1 μg / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, and 20 ng / mL, respectively, and place it in a 4°C refrigerator for use.

[0143] ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC) ELution conditions were optimized for the mixed solution of seven reference compounds to obtain the best separation.

[0144] Among them, the ultra-high performance liquid phase conditions are as follows: Agilent ZORBAX Eclipse Plμs C18 column, 1.8μm, 2.1×150mm, detection wavelength: 240nm; column temperature: 35℃; sample volume: 2μL; flow rate: 0.4mL / min; mobile phase A: 0.01% formic acid; mobile phase B: acetonitrile; elution program: 0min: 80% A, 20% B; 12min: 75% A, 25% B; 14min: 63% A, 37% B; 19min: 55% A, 45% B; 38min: 10% A, 90% B; 40min: 0% A, 100% B.

[0145] Steps for obtaining compound mass spectrometry information: Seven compound reference substances were prepared into 10 ppm methanol solutions, and the parent ion information of the seven compounds was confirmed in negative ion mode. Then, the Agilent Optimizer software was used to automatically optimize the product ions and collision energies after confirming the parent ions.

[0146] Compound 1: retention time: 11.65 min; parent ion: 279.1; quantitative ion: 245.0, collision energy: 17; qualifier ion: 173.0, collision energy 21;

[0147] Compound 2: retention time: 19.98 min; parent ion: 345.1; quantitative ion: 300.9, collision energy: 13; qualifier ion: 121.0, collision energy 29;

[0148] Compound 3: retention time: 23.27 min; parent ion: 358.2; quantitative ion: 274.0, collision energy: 29; qualifier ion: 219.8, collision energy 33;

[0149] Compound 4: retention time: 23.89 min; parent ion: 314.2; quantitative ion: 229.9, collision energy: 29; qualifier ion: 175.9, collision energy 25;

[0150] Compound 5: retention time: 28.21 min; parent ion: 428.2; quantitative ion: 343.9, collision energy: 37; qualifier ion: 297.9, collision energy 37;

[0151] Compound 6: retention time: 28.87 min; parent ion: 414.2; quantitative ion: 340.1, collision energy: 25; qualifier ion: 256.0, collision energy 41;

[0152] Compound 7; retention time: 30.41 min; parent ion: 418.2; quantitative ion: 334.0, collision energy: 19; qualifier ion: 280.0, collision energy 33;

[0153] Establishment of a quantitative analysis method using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry: Under the ultra-high performance liquid chromatography and triple quadrupole mass spectrometry parameter settings in the data acquisition software (Agilent MassHμnter Data Acqμisition), the mass spectrometry acquisition mode was changed to multiple reaction monitoring (MRM). Then, the compound's parent ion information, quantitative and qualitative product ion pair information, collision energy and other information were imported into the detection method. A mixed reference solution of seven compounds was sampled, and after running the program, the acquisition mode was updated to dynamic multiple reaction monitoring (DMRM).

[0154] Among them, the chromatographic conditions for ultra-performance liquid chromatography separation are as follows: an Agilent ZORBAX Eclipse PlμsC18 column, 1.8μm, 2.1×150mm, detection wavelength: 240nm; column temperature: 35°C; sample volume: 2μL; flow rate: 0.4mL / min; mobile phase A: 0.01% formic acid; mobile phase B: acetonitrile; elution program: 0min: 80% A, 20% B; 12min: 75% A, 25% B; 14min: 63% A, 37% B; 19min: 55% A, 45% B; 38min: 10% A, 90% B; 40min: 0% A, 100% B.

[0155] Among them, the triple quadrupole mass spectrometry analysis conditions were: electrospray ionization source AJS ESI was used as the ion source, detection was carried out in negative ion mode, dynamic multiple reaction monitoring DMRM was selected, the capillary voltage and outlet voltage were 3500 V and 380 V, respectively, the nozzle voltage was 2000 V, the dryer temperature and flow rate were 200 °C and 16 L / min, respectively, and the sheath gas temperature and flow rate were 350 °C and 12 L / min, respectively.

[0156] Limit of Detection and Limit of Quantitation: The limit of detection (LOD) and limit of quantitation (LOQ) were calculated based on the standard deviation of the response and the slope of the standard curve. Where: LOD = 3σ / S, LOQ = 10σ / S, σ: standard deviation of the response, S: slope of the standard curve, and the standard deviation of the response is the residual standard deviation of the standard curve.

[0157] Preparation of standard curve: Prepared mixed reference solutions at 5 μg / mL, 2 μg / mL, 1 μg / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, and 20 ng / mL were loaded onto the four optimized ultra-high performance liquid chromatography (UPLC) separation conditions and triple quadrupole mass spectrometry (QPS) analysis conditions described above. The quantitative standard curve equation was prepared with the compound concentration plotted on the abscissa and the quantitative ion response value plotted on the ordinate. See Table 1 below.

[0158] Table 1. Linear range, linear equation, correlation coefficient, detection limit and quantification limit of 7 compounds

[0159]

[0160] Example 5 Verification of the detection method of Example 4

[0161] The method validation refers to the laboratory quality control specifications, food physical and chemical testing standards and relevant provisions of the pharmacopoeia.

[0162] Precision: Intra-day precision was calculated by injecting the mixed standard solution six times on the same day. The concentrations of the seven compounds obtained from these six experiments were calculated based on the standard curve. Inter-day precision was calculated based on the results of nine experiments with three injections of the mixed standard solution daily for three consecutive days. The results showed that the RSDs for the intra-day precision of the seven compounds were 0.53%, 0.81%, 0.82%, 0.98%, 0.40%, 0.54%, and 0.7%, respectively. The RSDs for the inter-day precision were 1.35%, 2.07%, 2.13%, 1.82%, 1.56%, 1.84%, and 1.25%, respectively, all less than 15.00%, demonstrating good intra-day and inter-day precision of this method. Detailed results are shown in Tables 2 and 3.

[0163] Stability: Dried mycelium of strain 1 was extracted according to the procedure in Example 1. Sample solutions were injected at 0, 2, 4, 6, 8, 12, and 24 hours. Sample stability was calculated based on the results of seven experiments. The RSDs for the seven compounds were 2.86%, 0.60%, 1.23%, 0.93%, 1.64%, 0.81%, and 0.82%, respectively, all less than 15.00%, indicating that the sample was stable over a 24-hour period. Detailed results are shown in Table 4.

[0164] Repeatability: Six replicates of dried mycelium from strain 1 were weighed and processed according to the extraction procedure described in Example 2. The sample solution was then injected into the sample. Sample repeatability was calculated based on the six experimental results. The RSDs for the seven compounds were 0.49%, 0.88%, 1.90%, 1.37%, 3.01%, 3.08%, and 1.37%, respectively, all less than 15.00%, indicating good sample repeatability. Detailed results are shown in Table 5.

[0165] Sample recovery rate: Take a sample solution of known concentration, add the compound reference substance, mix well and repeat the sample loading three times to calculate the sample recovery rate.

[0166] Recovery rate of sample addition % = (measured value - amount of the measured component contained in the test sample) / amount of reference substance added × 100%

[0167] The specific results of sample recovery are shown in Table 6. The results showed that the RSDs of the seven compounds were all within 15%, which met the method requirements.

[0168] Example 6 Detection of 7 compounds in samples

[0169] Extraction of Hericium erinaceus mycelium: The Hericium erinaceus strain was 7080 (strain 1), and the fermentation method was the same as in Example 1. Weigh 0.50 g of dried Hericium erinaceus mycelium, add 10 mL of analytical-grade ethanol at a solid-liquid ratio of 1:20 (weight to volume, g: ml), and ultrasonically extract for 60 min. The supernatant was filtered through a 0.22 μm pore size organic phase microporous membrane to obtain the sample solution to be tested.

[0170] Source of Hericium erinaceus fruiting bodies: Hericium erinaceus fruiting bodies were collected from various provinces in China, including HE-1 (Shaoguan, Guangdong), HE-2 (Bijie, Guizhou), HE-3 (Xiangyang, Hubei), HE-4 (Huangshan, Anhui), HE-5 (Lishui, Zhejiang), HE-6 (Jinggangshan, Jiangxi), HE-7 (Lishui, Sichuan), HE-8 (Kunming, Yunnan), HE-9 (Hailin, Heilongjiang), HE-10 (Chifeng, Inner Mongolia), and HE-11 (Baishan, Jilin).

[0171] Extraction of Hericium erinaceus fruiting bodies: Weigh 0.50 g of each dried and crushed Hericium erinaceus fruiting body, add 10 mL of ethanol at a solid-liquid ratio of 1:20 (weight-to-volume ratio, weight g: volume ml), and extract by ultrasonication for 60 min. Take the supernatant and filter it through an organic phase microporous filter membrane with a pore size of 0.22 μm to obtain the sample solution to be tested.

[0172] Test results: The Hericium erinaceus mycelium test sample solution (strain 1) and the Hericium erinaceus fruiting body test sample solution were tested according to the detection method of Example 4. The specific test results are shown in Tables 7 and 8.

[0173] Table 2. Intra-day precision results

[0174]

[0175] Table 3. Inter-day precision results

[0176]

[0177] Table 4. Sample stability results

[0178]

[0179] Table 5. Sample repeatability results

[0180]

[0181] Table 6. Sample recovery results

[0182]

[0183] Table 7. Mycelium determination results

[0184]

[0185] Table 8. Quantitative results of Hericium erinaceus fruiting bodies from different origins

[0186]

[0187] Table 9 IC of compounds inhibiting α-glucosidase 50 value

[0188]

Claims

1. A method for simultaneously detecting seven compounds in Hericium erinaceus, characterized in that The method comprises the following steps: (1) Pretreatment steps of Hericium erinaceus samples: add Hericium erinaceus fruiting bodies or mycelium to ethanol for ultrasonic extraction or heating extraction at 60°C, take the supernatant, filter, and the obtained filtrate is the sample solution to be tested; prepare a mixed reference solution of 7 compounds; (2) Ultra-high performance liquid chromatography detection step: The sample solution and the mixed reference solution of the seven compounds were separated by ultra-high performance liquid chromatography to obtain a good separation degree; (3) Compound mass spectrometry information acquisition step: Using the mass spectrometry optimization software Agilent Optimizer, the seven compounds were scanned for parent ions, detected for daughter ion pairs, and searched for the optimal collision energy; (4) Steps for establishing the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry analytical method: In the data acquisition software Agilent MassHunter Data Acquisition, under ultra-high performance liquid chromatography analysis conditions and triple quadrupole mass spectrometry detection conditions, retention time, parent ion, daughter ion pair, and collision energy data were imported to establish a dynamic multi-reaction DMRM monitoring and analytical determination method for the seven compounds; at the same time, mixed reference solutions and test sample solutions of the seven compounds were loaded for determination; (5) Data analysis step: using the quantitative analysis software Agilent MassHunter Quantitative Analysis, standard curves of seven compounds were created to validate the established method and quantitatively analyze the sample solutions; The chromatographic conditions for ultra-high performance liquid chromatography separation in step (2) are: Agilent ZORBAX Eclipse Plus C18 column, 1.8 μm, 2.1 × 150 mm; column temperature: 35 °C; sample volume: 2 μL; flow rate: 0.4mL / min; mobile phase A: 0.01% formic acid; mobile phase B: acetonitrile; elution program 0min: 80% A, 20% B; 12min: 75% A, 25% B; 14min: 63% A, 37% B; 19min: 55% A, 45% B; 38min: 10% A, 90% B; 40min: 0% A, 100% B; Wherein, in step (4), ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used as the analytical detection instrument, wherein the chromatographic conditions of ultra-high performance liquid chromatography separation are: Agilent ZORBAX Eclipse Plus C18 chromatographic column, 1.8 μm, 2.1×150 mm; column temperature: 35° C.; sample volume: 2 μL; flow rate: 0.4 mL / min; mobile phase A: 0.01% formic acid; mobile phase B: acetonitrile; elution program: 0 min: 80% A, 20% B; 12 min: 75% A, 25% B; 14min: 63% A, 37% B; 19min: 55% A, 45% B; 38min: 10% A, 90% B; 40min: 0% A, 100% B; The seven compounds described are:

2. The method for simultaneously detecting seven compounds in Hericium erinaceus according to claim 1, wherein the pretreatment step of the Hericium erinaceus mycelium or fruiting body sample in step (1) is as follows: freeze-drying the sample to be tested, adding ethanol at a solid-liquid ratio of 1:20-1:40, weight g: volume ml, ultrasonic extraction for 10-90 min or heating extraction at 60°C for 10-90 min, and filtering the extract with an organic phase microporous filter membrane with a pore size of 0.22 μm to obtain a sample solution to be tested.

3. The method for simultaneously detecting 7 compounds in Hericium erinaceus according to claim 1, wherein the preparation method of the mixed reference solution of the 7 compounds in step (1) is: taking 7 compound reference substances, preparing a mixed standard solution with mass spectrometry grade methanol at a concentration of 5 μg / mL, and then diluting stepwise to obtain mixed standard working solutions of 2 μg / mL, 1 μg / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, and 20 ng / mL.

4. The method for simultaneous detection of seven compounds in Hericium erinaceus according to claim 1, wherein the retention time, parent ion, daughter ion pair, and collision energy information of each compound required for analysis in step (3) are obtained using mass spectrometry optimization software Agilent Optimizer as follows: Erinacerin X: retention time: 11.65 min; parent ion: 279.1; quantitative ion: 245.0, collision energy: 17; qualifier ion: 173.0, collision energy 21; Corallocin A: retention time: 19.98 min; parent ion: 345.1; quantitative ion: 300.9, collision energy: 13; qualifier ion: 121.0, collision energy 29; Hericerin A: retention time: 23.27 min; parent ion: 358.2; quantitative ion: 274.0, collision energy: 29; qualifier ion: 219.8, collision energy 33; N-De phenylethyl isohericerin: retention time: 23.89 min; parent ion: 314.2; quantitative ion: 229.9, collision energy: 29; qualifier ion: 175.9, collision energy 25; Erinacerin Y: retention time: 28.21 min; parent ion: 428.2; quantitative ion: 343.9, collision energy: 37; qualifier ion: 297.9, collision energy 37; Erinacerin Z: retention time: 28.87 min; parent ion: 414.2; quantitative ion: 340.1, collision energy: 25; qualifier ion: 256.0, collision energy 41; Isohericerin; retention time: 30.41 min; parent ion: 418.2; quantitative ion: 334.0, collision energy: 19; qualifier ion: 280.0, collision energy 33.

5. The method for simultaneously detecting seven compounds in Hericium erinaceus according to claim 1, wherein in step (4), ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used as the analytical detection instrument, wherein the mass spectrometry conditions are: electrospray ionization source AJSESI is used as the ion source, detection is carried out in negative ion mode, dynamic multiple reaction monitoring DMRM is selected, the capillary voltage is 3500V, the capillary outlet voltage is 380V, the nozzle voltage is 2000V, the dryer temperature and flow rate are 200°C and 16L / min respectively, and the sheath gas temperature and flow rate are 350°C and 12L / min respectively.

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