Edible mushroom glycoprotein with ACE / DPP-IV / TPP II triple inhibitory activity as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biochemistry, in particular to edible mushroom glycoprotein with ACE / DPP-IV / TPP II triple inhibitory activity, a preparation method and application. The invention relates to an edible mushroom glycoprotein with ACE / DPP-IV / TPP II triple inhibitory activity. The edible mushroom glycoprotein is a glycoprotein molecule with 23 glycosylation sites on an NADH-ubiquinone oxidoreductase chain 4; in the 23 glycosylation sites, sugar molecules modified on 11 glycosylation sites are fructose, and sugar molecules modified on 12 glycosylation sites are acetylated glucose; the 11 glycosylation sites are Ser21, Ser202, Ser433, Ser439, Ser486, Ser489, Ser503, Ser506, Ser516, Thr519 and Thr524, and the 12 glycosylation sites are Ser18, Ser26, Ser28, Thr37, Ser56, Ser70, Ser184, Ser202, Asn484, Ser490, Ser496 and Thr555. The invention further discloses a method for preparing the glycosylation kit. The edible mushroom glycoprotein has ACE inhibitory activity, DPP-IV inhibitory activity and TPP II inhibitory activity at the same time, and can be used for multichannel coordinated regulation of metabolic syndrome.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and particularly relates to an edible mushroom glycoprotein with triple inhibitory activities of ACE / DPP-IV / TPP II, a preparation method and applications thereof. Background Art

[0002] Metabolic syndromes (such as hypertension and hyperglycemia) and tumors have become the main threats to public health. Angiotensin-converting enzyme (ACE), dipeptidyl peptidase-IV (DPP-IV) and tripeptidyl peptidase II (TPP II) are respectively key targets for regulating blood pressure, blood glucose homeostasis and tumor microenvironment. ACE catalyzes the conversion of angiotensin I to angiotensin II and is a classical target for hypertension treatment; DPP-IV degrades incretin hormones (such as GLP-1), and inhibiting its activity can improve insulin resistance; TPP II is involved in antigen processing and cell cycle regulation, and its overexpression is related to tumor immune escape and chemoresistance. Although existing drugs (such as ACE inhibitors and DPP-IV inhibitors) have certain curative effects, most of them are single-target interventions, which are difficult to meet the requirements of multi-pathway coordinated regulation of metabolic syndromes, and long-term use may be accompanied by side effects (such as dry cough and hypoglycemia risk). In addition, the research and development of anti-tumor drugs targeting TPPII is still in the early stage, and natural multi-target inhibitors are urgently needed to be developed.

[0003] Edible mushrooms are rich in glycoprotein components. Existing studies have confirmed that glycoprotein components have immunomodulatory, antioxidant and anti-tumor activities, but there is still a blank in the multi-target inhibition research of key metabolic enzymes. Existing studies mostly focus on single-enzyme inhibition (such as ACE or DPP-IV), lacking the exploration of coordinated regulation of the ACE / DPP-IV / TPP II triple pathway; the activity of glycoproteins is closely related to their sugar chain structure and glycopeptide linkage mode, but glycoprotein molecules with definite multi-target inhibitory activities have not been analyzed, and the structure-function relationship is unclear; edible mushroom glycoproteins are mostly used as ordinary dietary supplements and are not deeply combined with products for precise health intervention, so the application scenarios are narrow. Summary of the Invention

[0004] In view of the above technical problems, the present invention provides an edible mushroom glycoprotein with triple inhibitory activities of ACE / DPP-IV / TPP II, a preparation method and applications thereof. The present invention prepares a glycoprotein with triple inhibitory activities of ACE / DPP-IV / TPP II from Stropharia rugosoannulata, and through structure analysis and function verification, proposes its innovative applications in antihypertensive, hypoglycemic and anti-tumor products.

[0005] The technical solution of the present invention is as follows:

[0006] The present invention provides an edible mushroom glycoprotein with triple inhibitory activities against ACE / DPP-IV / TPP II. The edible mushroom glycoprotein is a glycoprotein molecule having 23 glycosylation sites on the NADH-ubiquinone oxidoreductase chain 4; among the 23 glycosylation sites, the sugar molecules modified on 11 glycosylation sites are fructose, and the sugar molecules modified on 12 glycosylation sites are acetylated glucose; the 11 glycosylation sites are Ser21, Ser202, Ser433, Ser439, Ser486, Ser489, Ser503, Ser506, Ser516, Thr519, Thr524, and the 12 glycosylation sites are Ser18, Ser26, Ser28, Thr37, Ser56, Ser70, Ser184, Ser202, Asn484, Ser490, Ser496, Thr555.

[0007] The present invention also provides a method for preparing the edible mushroom glycoprotein, which includes mixing the powder of Stropharia rugosoannulata with water, performing four-frequency ultrasonic extraction with the ultrasonic frequency combination of 23 + 25 + 28 + 40 KHz, then filtering, collecting the supernatant, and freeze-drying.

[0008] As an embodiment, the particle size of the Stropharia rugosoannulata powder is less than 100 mesh.

[0009] As an embodiment, the material-liquid ratio of the mixing is 1 g: 10 - 30 mL.

[0010] As an embodiment, the material-liquid ratio of the mixing is 1 g: 20 - 30 mL.

[0011] As an embodiment, the four-frequency ultrasonic is slit four-frequency ultrasonic. The slit means that the diameter of the treatment cavity is not more than 5 cm.

[0012] As an embodiment, the power density of the slit four-frequency ultrasonic is 100 - 200 W / L.

[0013] As an embodiment, the alternating working time of each frequency of the slit four-frequency ultrasonic is 1.5 - 5 s.

[0014] As an embodiment, the ultrasonic intermittent ratio is 6:2 - 20:2.

[0015] The present invention also provides an application of the edible mushroom glycoprotein or the preparation method in products with triple inhibitory activities against ACE / DPP-IV / TPPII.

[0016] Compared with the prior art, the beneficial effects of the present invention:

[0017] 1. The molecular weight of the edible mushroom glycoprotein of the present invention is 65884.14 Da, and it is a glycoprotein molecule with 23 glycosylation sites on NADH-ubiquinone oxidoreductase chain 4. Among them, among the 23 glycosylation sites, the sugar molecules modified on 11 glycosylation sites are fructose molecules, and the sugar molecules modified on 12 glycosylation sites are acetylated glucose. The 11 glycosylation sites are Ser21, Ser202, Ser433, Ser439, Ser486, Ser489, Ser503, Ser506, Ser516, Thr519, Thr524, and the 12 glycosylation sites are Ser18, Ser26, Ser28, Thr37, Ser56, Ser70, Ser184, Ser202, Asn484, Ser490, Ser496, Thr555. The edible mushroom glycoprotein contains 314 transmembrane regions, and both the N-terminus and the C-terminus are located outside the membrane.

[0018] 2. The edible mushroom glycoprotein of the present invention has a strong binding force to the ACE receptor protein, can significantly inhibit the activity of the ACE receptor protein, and has potential antihypertensive activity. The edible mushroom glycoprotein has a strong binding force to the DPP-IV receptor protein, can significantly inhibit the activity of the DPP-IV receptor protein, and has potential hypoglycemic activity. The edible mushroom glycoprotein has a strong binding force to the TPP II receptor protein, can significantly inhibit the activity of the TPP II receptor protein, and has potential anti-tumor activity. It shows that the edible mushroom glycoprotein of the present invention simultaneously has ACE inhibitory activity, DPP-IV inhibitory activity and TPP II inhibitory activity, and can be used for multi-pathway synergistic regulation of metabolic syndrome. Description of the Drawings

[0019] Figure 1 is the amino acid sequence of the glycoprotein of Stropharia rugosoannulata;

[0020] Figure 2 is the transmembrane region of the protein of Stropharia rugosoannulata analyzed by TMHMM;

[0021] Figure 3 is the spatial structure of the glycoprotein of Stropharia rugosoannulata; among them, the protein of Stropharia rugosoannulata is represented in a ribbon model (red), and the bound sugar molecules are represented in a ball-and-stick model (green);

[0022] Figure 4 is the binding diagram of the ACE receptor protein and the glycoprotein molecule of Stropharia rugosoannulata; among them, A is the 3D binding diagram of the ACE receptor protein and the glycoprotein molecule of Stropharia rugosoannulata. The ACE receptor protein and the protein of Stropharia rugosoannulata are represented in a surface model, and the sugar molecules bound to the protein of Stropharia rugosoannulata are represented in a ball-and-stick model. White is the ACE receptor protein, pink is the protein of Stropharia rugosoannulata, and green is the sugar molecule; B is the binding energy diagram of the ACE receptor protein and the glycoprotein molecule of Stropharia rugosoannulata;

[0023] Figure 5 It is a binding diagram of DPP-IV receptor protein and Stropharia rugosoannulata glycoprotein molecule. Among them, A is a 3D diagram of the binding of DPP-IV receptor protein and Stropharia rugosoannulata glycoprotein molecule. The DPP-IV receptor protein and Stropharia rugosoannulata protein are represented in surface mode, and the sugar molecules bound to the Stropharia rugosoannulata protein are represented in ball-and-stick mode. White is the DPP-IV receptor protein, orange is the Stropharia rugosoannulata protein, and green is the sugar molecule; B is the energy diagram of the binding of DPP-IV receptor protein and Stropharia rugosoannulata glycoprotein molecule.

[0024] Figure 6 It is a binding diagram of TPP II receptor protein and Stropharia rugosoannulata glycoprotein molecule. Among them, A is a 3D diagram of the binding of TPP II receptor protein and Stropharia rugosoannulata glycoprotein molecule. The TPP II receptor protein and Stropharia rugosoannulata protein are represented in surface mode, and the sugar molecules bound to the Stropharia rugosoannulata protein are represented in ball-and-stick mode. White is the TPP II receptor protein, yellow is the Stropharia rugosoannulata protein, and green is the sugar molecule; B is the energy diagram of the binding of TPP II receptor protein and Stropharia rugosoannulata glycoprotein molecule. Detailed implementation mode

[0025] The present invention provides an edible mushroom glycoprotein with triple inhibitory activity against ACE / DPP-IV / TPP II. The present invention uses Spectronaut TM software to perform qualitative and quantitative analysis on this edible mushroom glycoprotein according to the spectral library. After mass spectrometry identification, the glycoprotein molecule is NADH-ubiquinone oxidoreductase (the numbering information of the NADH-ubiquinone oxidoreductase protein molecule in UniProt (https: / / www.uniprot.org) is A0A650AG65_9PEZI, and its amino acid sequence is as shown in SEQ.ID No.1 and Figure 1An edible mushroom glycoprotein having 23 glycosylation sites on chain 4 (as shown). In the present invention, the edible mushroom glycoprotein is a glycoprotein having 23 glycosylation sites on the NADH-ubiquinone oxidoreductase chain 4. Among the 23 glycosylation sites, the sugar molecules modified on 11 glycosylation sites are fructose, and the sugar molecules modified on 12 glycosylation sites are acetylated glucose. The 11 glycosylation sites are Ser21, Ser202, Ser433, Ser439, Ser486, Ser489, Ser503, Ser506, Ser516, Thr519, Thr524 respectively, and the 12 glycosylation sites are Ser18, Ser26, Ser28, Thr37, Ser56, Ser70, Ser184, Ser202, Asn484, Ser490, Ser496, Thr555 respectively. In the present invention, the TMHMM-2.0 online tool is used to analyze the transmembrane structure of the edible mushroom glycoprotein. The glycoprotein contains 314 transmembrane regions, and both the N-terminus and the C-terminus are located outside the membrane. The present invention also uses the MOE software to perform glycosylation modification of the protein sequence according to the glycosylation sites identified by mass spectrometry to obtain the edible mushroom glycoprotein molecule and its spatial structure, see Figure 3 .

[0026] The present invention also provides a preparation method of the edible mushroom glycoprotein. The preparation method includes mixing the powder of Stropharia rugosoannulata with water, performing four-frequency ultrasonic extraction, filtering, collecting the supernatant, and freeze-drying.

[0027] In the present invention, the Stropharia rugosoannulata is first dried and then crushed. As an implementation manner, the drying temperature is 50-70 °C, and the drying time is 5-24 h. The water content of the dried Stropharia rugosoannulata in the present invention is preferably not higher than 10%, which is beneficial to subsequent crushing into uniform fine powder. After crushing in the present invention, it is sieved through a 100-mesh sieve to promote the dissolution of the effective substances in Stropharia rugosoannulata in water. The present invention does not make special limitations on the crushing method, and commonly used ball milling and hammer milling in the art can be adopted. The present invention does not have special limitations on the specific source of the Stropharia rugosoannulata, and commercially available products can be used.

[0028] In the present invention, the powder of Stropharia rugosoannulata is mixed with water and subjected to four-frequency ultrasound. The four-frequency ultrasound generates ultrasonic waves of different frequencies, which can form a more complex and uniform cavitation field in the aqueous solution, increase the local shear force, facilitate the breaking of cell walls and cell membranes, and can also reduce the damage to heat-sensitive and easily oxidized components to a certain extent. In the present invention, the material-liquid ratio of the mixing is 1 g: 10 - 30 mL, preferably 1 g: 20 - 30 mL, and more preferably the material-liquid ratio is 1 g: 20 mL. The four-frequency ultrasound frequency is 23 + 25 + 28 + 40 kHz; in one embodiment, the four-frequency ultrasound is preferably slit four-frequency ultrasound. The slit four-frequency ultrasound means that the diameter of the four-frequency ultrasound treatment cavity (the diameter of the circular cross-section of the internal space of the cavity) is not greater than 5 cm. The alternating working time of each frequency of the slit four-frequency ultrasound is 1.5 - 5 s, preferably 1.5 s; the power density of the slit four-frequency ultrasound is 100 - 200 W / L, preferably 100 W / L; the time of the slit four-frequency ultrasound is 20 - 60 min, preferably 30 min; the intermittent ratio of the slit four-frequency ultrasound is 6:2 - 20:2, preferably 6:2. The parameters of the slit four-frequency ultrasound in the present invention can be used to extract the edible mushroom glycoprotein. The parameters of the slit four-frequency ultrasound in the present invention can have different effects on components with different properties, so as to achieve selective extraction, improve the extraction efficiency of the edible mushroom glycoprotein, and protect the structure and function of the edible mushroom glycoprotein.

[0029] In the present invention, after the ultrasonic treatment is completed, the supernatant is filtered, collected, and freeze-dried. In the present invention, no special limitation is imposed on the filtering method, and common pressure filtration, vacuum filtration, and centrifugal filtration in the art can be used. In the examples of the present invention, the supernatant is collected by centrifugal filtration, preferably centrifuged at 1000 - 8000 rpm for 15 - 60 min, and more preferably centrifuged at 8000 rpm for 15 min. In the present invention, the supernatant is freeze-dried to obtain the freeze-dried powder of Stropharia rugosoannulata, and no special limitation is imposed on the freeze-drying method, and common pressure freeze-drying and vacuum freeze-drying in the art can be used. In the examples of the present invention, the freeze-drying is carried out by low-temperature freeze-drying, the temperature of the low-temperature freeze-drying is -20 °C to -70 °C, preferably -70 °C; the time of the low-temperature freeze-drying is not less than 48 h, preferably 48 h. The low-temperature freeze-drying in the present invention can effectively reduce the content of residual moisture and improve the stability of the edible mushroom glycoprotein. The present invention further verifies that the edible mushroom glycoprotein has a strong binding force to the ACE receptor protein, can significantly inhibit the activity of the ACE receptor protein, and has potential antihypertensive activity; has a strong binding force to the DPP-IV receptor protein, can significantly inhibit the activity of the DPP-IV receptor protein, and has potential hypoglycemic activity; has a strong binding force to the TPP II receptor protein, can significantly inhibit the activity of the TPP II receptor protein, and has potential antitumor activity.

[0030] The present invention also provides an application of the edible mushroom glycoprotein or the preparation method in products with triple inhibitory activities of ACE / DPP-IV / TPP II. By simultaneously exerting the ACE inhibitory activity, DPP-IV inhibitory activity, and TPP II inhibitory activity, multi-pathway synergistic regulation of metabolic syndrome is achieved.

[0031] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be described in detail below in conjunction with embodiments. However, they should not be construed as limiting the protection scope of the present invention.

[0032] For the materials, reagents, etc. used in the following embodiments, unless otherwise specified, the reagents, consumables, etc. involved in the present invention can be obtained from commercial channels. If the specific usage conditions are not indicated, they are usually carried out under conventional conditions or according to the conditions recommended by the company.

[0033] Example 1

[0034] Using Stropharia rugosoannulata as the raw material, it was dried at 50 °C for 8 h to make the water content of Stropharia rugosoannulata constant (water content < 10%), and then ball-milled and sieved to obtain Stropharia rugosoannulata powder with a particle size less than 100 mesh. The Stropharia rugosoannulata powder was mixed with water at a solid-liquid ratio of 1:20 (g / mL), and glycoprotein was extracted by slit four-frequency ultrasound. The ultrasound frequency combination was 23 + 25 + 28 + 40 (kHz), the ultrasound intermittent ratio was 6:2 s / s (ultrasound working for 6 s and stopping for 2 s), the alternating working time of each frequency in the four frequencies was 1.5 s, the ultrasound power density was 100 W / L. After 30 min of ultrasound, it was centrifuged at 8000 rpm for 15 min, and the supernatant was collected and freeze-dried at -70 °C for 48 h to obtain Stropharia rugosoannulata freeze-dried powder.

[0035] Example 2

[0036] Using Stropharia rugosoannulata as the raw material, it was dried at 50 °C for 8 h to make the water content of Stropharia rugosoannulata constant (water content < 10%), and then ball-milled and sieved to obtain Stropharia rugosoannulata powder with a particle size less than 100 mesh. The Stropharia rugosoannulata powder was mixed with water at a solid-liquid ratio of 1:30 (g / mL), and glycoprotein was extracted by slit four-frequency ultrasound. The ultrasound frequency combination was 23 + 25 + 28 + 40 (kHz), the ultrasound intermittent ratio was 20:2 s / s (ultrasound working for 6 s and stopping for 2 s), the alternating working time of each frequency in the four frequencies was 5 s, the ultrasound power density was 200 W / L. After 30 min of ultrasound, it was centrifuged at 8000 rpm for 15 min, and the supernatant was collected and freeze-dried at -70 °C for 48 h to obtain Stropharia rugosoannulata freeze-dried powder.

[0037] Example 3 Identification and Structural Analysis of Edible Mushroom Glycoprotein Sequence

[0038] 1. Identification of Edible Mushroom Glycoprotein Sequence

[0039] Take 100 μg of the lyophilized powder sample of Stropharia rugosoannulata obtained in Example 1, and dissolve the sample thoroughly with 100 μL of 100 mM triethylammonium bicarbonate buffer (TEAB). Then add tris(2-carboxyethyl)phosphine (TCEP) to make the final concentration of TCEP 10 mM, and react at 37 °C for 60 min. Then add iodoacetamide (IAM) to make the final concentration of IAM 40 mM, and react in the dark at room temperature for 40 min. Finally, centrifuge at 10000 g for 20 min to obtain the protein precipitate. Add trypsin to the protein precipitate according to a mass ratio of 1:50, and digest at 37 °C overnight. After trypsin digestion, take an equal amount of the sample and dry the peptide segments in a vacuum centrifugal concentrator; redissolve the dried peptide segments after enzymatic digestion with 0.1% trifluoroacetic acid (TFA) and then desalt, and dry with a vacuum concentrator; use NANO DROP ONE (Thermo Scientific) to perform peptide quantification by ultraviolet spectrophotometry. Dissolve an equal amount of the peptide segments with the mass spectrometry loading buffer and perform data-independent mass spectrometry acquisition mode DIA detection and analysis. Analytical column: uPAC High Throughptu column (75 μm × 5.5 cm, Thermo, USA), chromatograph: Vanquish Neo (Thermo, USA), mass spectrometer: Astral (Thermo, USA), chromatographic separation time: 8 min, mobile phase A: 2% acetonitrile 0.1% formic acid, mobile phase B: 80% acetonitrile 0.1% formic acid, liquid phase elution gradient: 0 min, 4% B; 0.1 min, 8% B; 1 min, 12.5% B; 1.1 min, 12.6% B; 3.6 min, 22.5% B; 5.8 min, 45% B; 6.4 min, 99% B; 8 min, 99% B. The detection mode is positive ion, the ion source voltage is set to 1.5 kV, and the mass spectrometry scanning range is set to 100 - 1700 m / z. Use Spectronaut TM The Spectronaut software was used to perform qualitative and quantitative analysis of glycoproteins in the samples according to the spectral library.

[0040] After mass spectrometry identification, 1 glycoprotein molecule with 23 glycosylation sites was obtained in the lyophilized powder of Stropharia rugosoannulata, with a molecular weight of 65884.14 Da. In this glycoprotein molecule, the protein molecule has the accession number A0A650AG65_9PEZI in UniProt (https: / / www.uniprot.org) and is NADH-ubiquinone oxidoreductase chain 4; among the 23 glycosylation sites, the sugar molecules modified at 11 glycosylation sites are fructose molecules, and the sugar molecules modified at 12 glycosylation sites are acetylated glucose. Among them, the amino acid information of fructose and acetylated glucose molecules linked to the protein molecule is shown in Table 1, and the glycoprotein sequence is as Figure 1 shown.

[0041] Table 1 Information on glycosylation sites of edible mushroom glycoproteins identified

[0042] Glycosylation site Sugar molecule Glycosylation site Sugar molecule Ser489 Fructose Ser202 N-acetylglucosamine Ser486 Fructose Asn484 N-acetylglucosamine Ser503 Fructose Ser26 N-acetylglucosamine Ser516 Fructose Ser70 N-acetylglucosamine Ser439 Fructose Ser490 N-acetylglucosamine Ser21 Fructose Ser56 N-acetylglucosamine Ser433 Fructose Ser496 N-acetylglucosamine Thr519 Fructose Ser18 N-acetylglucosamine Ser506 Fructose Ser28 N-acetylglucosamine Thr524 Fructose Thr37 N-acetylglucosamine Ser202 Fructose Thr555 N-acetylglucosamine Ser184 N-acetylglucosamine

[0043] 2. Structural analysis of edible mushroom glycoproteins

[0044] TMHMM-2.0 is an online tool for predicting transmembrane helices in proteins. The TMHMM-2.0 tool is based on deep learning algorithms and can analyze the amino acid sequence of proteins and predict the possible transmembrane helix regions therein, which is of great significance for understanding the structure and function of proteins and related research. TMHMM-2.0 was used to analyze the transmembrane regions of edible mushroom glycoproteins. By accessing the TMHMM-2.0 website server (https: / / services.healthtech.dtu.dk / services / TMHM M-2.0 / ), the amino acid sequence of the glycoprotein was uploaded to the server in the form of a FASTA format file, and the transmembrane structure of the glycoprotein was analyzed through the output of TMHM M results. As Figure 2 shown, the edible mushroom glycoprotein analyzed by TMHMM contains 314 transmembrane regions, and both the N-terminus and C-terminus are located outside the membrane.

[0045] The pdb structure file of Stropharia rugosoannulata protein A0A650AG65 was obtained from UniProt and uploaded to the MOE 2019 software (Chemical Computing Group ULC, Montreal, Canada). The MOE software performed glycosylation modification of the protein sequence according to the glycosylation sites identified by mass spectrometry to obtain the edible mushroom glycoprotein molecule and its spatial structure, as Figure 3 shown.

[0046] Example 4 Activity analysis of edible mushroom glycoproteins

[0047] 1. Evaluation of the antihypertensive effect of edible mushroom glycoproteins

[0048] Angiotensin-converting enzyme (ACE) plays a key role in inducing blood pressure elevation in the body's renin-angiotensin-aldosterone system and is a drug target for treating cardiovascular diseases such as hypertension and heart failure. The antihypertensive effect was evaluated by measuring the inhibitory activity of the freeze-dried powder of Stropharia rugosoannulata against ACE.

[0049] The analysis method used was the kit method (DOJINDO ACE Kit-WSTA502, Shanghai Yulu Biotechnology Co., Ltd.). Exactly 0.1 g of the freeze-dried powder of Stropharia rugosoannulata polysaccharide from Example 1 was weighed and dissolved in 50 mL of pure water, and then serially diluted to sample solutions with concentration gradients of 2.0 mg / mL, 0.4 mg / mL, 0.08 mg / mL, 0.016 mg / mL, 0.0032 mg / mL, and 0.00064 mg / mL. Take 20 μL of the sample solution, add 20 μL of the kit matrix buffer and 20 μL of the kit enzyme working solution, incubate at 37 °C for 60 min, add 200 μL of the kit indicator working solution, incubate at 25 °C for 10 min, and measure the absorbance at 450 nm. The analysis method was the same as that of the kit method.

[0050] Using the mass concentration of the sample solution and the inhibition activity value as the abscissa and ordinate, an inhibition curve was plotted, and from the inhibition curve, the mass concentration of the sample at 50% inhibition rate (IC 50 ) was calculated. The results of the in vitro ACE inhibition activity analysis showed that the IC 50 of the edible mushroom glycoprotein ACE inhibition was 0.128 mg / mL, indicating that the edible mushroom glycoprotein had good ACE inhibition activity.

[0051] 2. Evaluation of the hypoglycemic effect of edible mushroom glycoprotein

[0052] Dipeptidyl peptidase-IV (DPP-IV) is a serine protease widely distributed in the human body, which plays an important role in physiological processes such as glucose metabolism, immune regulation, and signal transduction, and has attracted much attention due to its close association with diabetes treatment. The hypoglycemic effect of the freeze-dried powder of Stropharia rugosoannulata was evaluated by measuring its inhibitory activity against DPP-IV.

[0053] The inhibitory activity of the freeze-dried powder of Stropharia rugosoannulata against DPP-IV was measured using the Abcam KA1311 DPP-IV inhibitor screening kit (Qingdao Qingke Sail Biotechnology Co., Ltd.). The DPP-IV inhibitor screening kit method is based on DPP-IV catalyzing the peptide bond in the fluorescent substrate Gly-Pro-Aminomethylcoumarin (AMC) to release free AMC groups, which can be detected by fluorescence at an excitation wavelength of 350 - 360 nm and an emission wavelength of 450 - 465 nm. By measuring the fluorescence intensity of AMC, the activity of DPP-IV can be measured. Subtracting the inhibited part from the initial activity of DPP-IV gives the DPP-IV inhibitor activity value (inhibition rate). Exactly 0.1 g of the freeze-dried powder of Stropharia rugosoannulata from Example 1 was weighed and dissolved in 50 mL of pure water, and then serially diluted to sample solutions with concentration gradients of 2.0 mg / mL, 0.4 mg / mL, 0.08 mg / mL, 0.016 mg / mL, and 0.0032 mg / mL. The analysis method was the same as that of the kit method.

[0054] Using the sample concentration and inhibition rate to plot an inhibition curve, calculate the concentration of the sample when the inhibition rate is 50% (IC 50 ). The results of in vitro DPP-IV inhibitory activity analysis showed that the IC 50 of edible mushroom glycoprotein against DPP-IV was 0.663 mg / mL, indicating that edible mushroom glycoprotein has good DPP-IV inhibitory activity.

[0055] 3. Evaluation of the anti-tumor effect of edible mushroom glycoprotein

[0056] Tripeptidyl peptidase II (TPP II) is a multifunctional lysosomal exoprotease widely present in eukaryotic cells. It is closely related to processes such as immune regulation, cell cycle regulation, and tumorigenesis, and has become one of the important targets in cancer treatment and metabolic disease research in recent years. The anti-tumor effect was evaluated by measuring the inhibitory activity of Lyophyllum decastes freeze-dried powder against TPP II.

[0057] The substrate Suc-AAPF-pNA can be hydrolyzed by TPP II to release pNA (p-nitroaniline), and the product has an absorption peak at 405 nm. By examining the change in absorbance of the product at 405 nm, the TPP II enzyme inhibitory activity of Lyophyllum decastes glycoprotein was calculated. The analysis method used a kit method (Tripeptidyl peptidase 2 (TPP2) detection kit, Shanghai Chaorui Biotechnology Co., Ltd.). Accurately weigh 0.1 g of the Lyophyllum decastes glycoprotein freeze-dried powder in Example 1, dissolve it in 50 mL of pure water, and serially dilute it to sample solutions with concentration gradients of 2.0 mg / mL, 0.4 mg / mL, 0.08 mg / mL, 0.016 mg / mL, 0.0032 mg / mL, and 0.00064 mg / mL. Take 20 μL of the sample solution, add 20 μL of the kit matrix buffer and 20 μL of the kit enzyme working solution, incubate at 37 °C for 15 min, add 120 μL of the kit substrate solution, react at 37 °C for 60 min, add 20 μL of the kit stop solution, and measure the absorbance at 405 nm.

[0058] Using the mass concentration of the sample solution and the inhibition activity value as the abscissa and ordinate to plot an inhibition curve, and calculate the mass concentration of the sample when the inhibition rate is 50% (IC 50 ). The results of in vitro TPP II inhibitory activity analysis showed that the IC 50 of edible mushroom glycoprotein against TPP II was 0.039 mg / mL, indicating that edible mushroom glycoprotein has good TPP II inhibitory activity.

[0059] Example 5 Molecular docking of functional target receptors and edible mushroom glycoprotein

[0060] Download the crystal structures of angiotensin-converting enzyme (PDB: 1O86), dipeptidyl peptidase-IV (PDB: 2ONC), and tripeptidyl peptidase II (PDB: 3LXU) from the RCSB database (https: / / www.rcsb.org / ). Optimize the crystal structures of the receptor proteins using MOE 2019 software to remove water molecules and complete hydrogen atoms. Perform molecular docking of the receptor proteins and the glycoprotein from Stropharia rugosoannulata using MOE software. Set the docking parameters as follows: select "receptor protein" for the receptor and "edible mushroom glycoprotein" for the ligand, and use the all-atom protein docking mode. Set the parameters for the molecular docking structure as "Pre-Placement, 10000; Placement, 1000; Refinement, 100". Analyze the binding scores and binding energies between the receptor proteins and the edible mushroom glycoproteins in the binding complexes using MOE software. The lower the binding scores and binding bond energies, the better the intermolecular binding effect.

[0061] The binding information of the ACE receptor protein and the edible mushroom glycoprotein in Example 1 is as Figure 4 shown. The results show that the binding score of the ACE receptor protein and the edible mushroom glycoprotein is -32.31, and the binding bond energy is -935.50 kcal / mol. The above results indicate that the edible mushroom glycoprotein has a strong binding affinity for the ACE receptor protein, can significantly inhibit the activity of the ACE receptor protein, and has potential antihypertensive activity.

[0062] The binding information of the DPP-IV receptor protein and the edible mushroom glycoprotein in Example 1 is as Figure 5 shown. The results show that the binding score of the DPP-IV receptor protein and the edible mushroom glycoprotein is -20.50, and the binding bond energy is -1222.68 kcal / mol. The above results indicate that the edible mushroom glycoprotein has a strong binding affinity for the DPP-IV receptor protein, can significantly inhibit the activity of the DPP-IV receptor protein, and has potential hypoglycemic activity.

[0063] The binding information of the TPP II receptor protein and the edible mushroom glycoprotein in Example 1 is as Figure 6 shown. The results show that the binding score of the TPP II receptor protein and the edible mushroom glycoprotein is -25.68, and the binding bond energy is -1821.79 kcal / mol. The above results indicate that the edible mushroom glycoprotein has a strong binding affinity for the TPP II receptor protein, can significantly inhibit the activity of the TPP II receptor protein, and has potential anti-tumor activity.

[0064] In summary, the edible mushroom glycoprotein described in the present invention has ACE inhibitory, DPP-IV inhibitory, and TPP II inhibitory activities, and can be applied to the development of antihypertensive, hypoglycemic, and anti-tumor products.

[0065] The above are only embodiments of the present invention, and thus do not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, shall similarly be included within the patent protection scope of the present invention.

Claims

1. An edible mushroom glycoprotein with triple inhibitory activities against ACE / DPP-IV / TPP II, characterized in that, The edible mushroom glycoprotein is a glycoprotein molecule with 23 glycosylation sites on the NADH-ubiquinone oxidoreductase chain 4; among the 23 glycosylation sites, the sugar molecules modified on 11 glycosylation sites are fructose, and the sugar molecules modified on 12 glycosylation sites are acetylated glucose; the 11 glycosylation sites are Ser21, Ser202, Ser433, Ser439, Ser486, Ser489, Ser503, Ser506, Ser516, Thr519, Thr524, and the 12 glycosylation sites are Ser18, Ser26, Ser28, Thr37, Ser56, Ser70, Ser184, Ser202, Asn484, Ser490, Ser496, Thr555.

2. The preparation method of the edible mushroom glycoprotein according to claim 1, characterized in that, The method includes mixing the powder of Stropharia rugosoannulata with water, performing four-frequency ultrasonic extraction with the ultrasonic frequency combination of 23 + 25 + 28 + 40 KHz, then filtering, collecting the supernatant, and freeze-drying.

3. The preparation method according to claim 2, characterized in that, The particle size of the powder of Stropharia rugosoannulata is less than 100 mesh.

4. The preparation method according to claim 2, characterized in that, The material-liquid ratio of the mixing is 1 g: 10 - 30 mL.

5. The preparation method according to claim 4, characterized in that, The material-liquid ratio of the mixing is 1 g: 20 - 30 mL.

6. The preparation method according to claim 2, characterized in that, The four-frequency ultrasound is slit four-frequency ultrasound.

7. The preparation method according to claim 6, characterized in that, The power density of the slit four-frequency ultrasound is 100 - 200 W / L.

8. The preparation method according to claim 6, wherein The alternating working time of each frequency of the slit four-frequency ultrasound is 1.5 - 5 s.

9. The preparation method according to claim 6, characterized in that, The ultrasonic intermittent ratio is 6:2 - 20:

2.

10. Use of the edible mushroom glycoprotein according to claim 1 or the preparation method according to any one of claims 2 - 9 in a product having triple inhibitory activity against ACE / DPP-IV / TPP II.

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