Anticancer active glycoprotein, its preparation method, application and anticancer drug for women

Abstract

The application belongs to the field of anticancer active natural ingredients, and particularly relates to an anticancer active glycoprotein, a preparation method and application thereof, and an anticancer drug for female cancer, wherein the anticancer active glycoprotein is a protein chemically bonded with selenium and polysaccharide, the protein includes a peroxidase domain protein, an RNA-dependent RNA polymerase, a nucleolar 278 pre-rRNA processing Urb / Npa2 C-terminal domain-containing protein, and a ribosomal protein S12; the polysaccharide chain is a sugar unit fragment with a structure of formula 1; the hydroxyl in the polysaccharide is connected to at least one amino acid in aspartic acid, valine, glutamic acid, arginine, lysine and serine in the protein through a hydrogen bond; and the selenium bond includes at least one of Se=O and Se-C-O. The application provides a novel structure of selenium glycoprotein ingredient, and further research shows that the ingredient can specifically inhibit female cancer cells and has a lower normal cell inhibition activity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the field of an anticancer active glycoprotein. Background Technology

[0002] Women's cancers (also known as female tumors) mainly include ovarian cancer, breast cancer, and cervical cancer. These cancers are insidious and difficult to detect, and have high mortality rates. For example, ovarian cancer has an insidious onset, with 70% of patients diagnosed at an advanced stage, and a five-year survival rate of less than 30%, urgently requiring targeted new drugs. Breast cancer is the most common cancer among women; the hormone receptor-positive subtype relies on endocrine therapy, and drug options are limited after drug resistance develops. Cervical cancer has a high incidence rate, and there is a lack of second-line treatment options for patients with advanced recurrence. Developing highly effective and low-toxicity anti-tumor drugs specifically for women can fill clinical gaps, prolong life, and improve quality of life. Therefore, researching anti-cancer drugs with specific killing capabilities against women's cancers and no toxic side effects on normal cells or tissues is urgently needed.

[0003] Natural plant glycoproteins target tumors gently and precisely by using sugars to lock onto their targets and proteins to kill them. Previous studies have shown that Ganoderma lucidum glycoproteins activate macrophages and can inhibit breast cancer metastasis; Lycium barbarum glycoproteins block PI3K / AKT and can induce apoptosis in ovarian cancer cells; Astragalus membranaceus glycoproteins enhance CD8... + T-cell infiltration can reverse drug resistance; it is low in toxicity, can be taken orally, and has a wide range of sources, providing a new approach for the highly effective and low-toxicity treatment of female cancers.

[0004] Changes in the polysaccharide and protein structures of glycoproteins can significantly affect their physiological activity. How to obtain glycoprotein components with novel structures that also possess excellent anti-cancer specificity for women remains an industry-wide challenge. Summary of the Invention

[0005] In view of the problems existing in the prior art, the primary objective of this invention is to provide an anti-cancer active glycoprotein (also referred to as Se-POGP-2b or POGP-2b in this invention), which aims to provide a novel glycoprotein that is adapted to the characteristics of female cancer cells and takes into account both excellent anti-cancer activity and specificity.

[0006] The second objective of this invention is to provide a method for preparing and applying the aforementioned anticancer active glycoprotein.

[0007] A third objective of this invention is to provide a medicament for treating female cancers comprising the aforementioned anticancer active glycoprotein.

[0008] Women's cancers mainly include ovarian cancer, breast cancer, and cervical cancer. The core characteristics of these cancers are insidious onset, atypical early symptoms, and a high correlation with hormones or HPV infection, requiring specialized screening for early detection. The challenges in treatment lie in early diagnosis; 70% of ovarian cancers are diagnosed at an advanced stage, and the proportion of late-stage cervical cancers is high in areas with low screening rates. Furthermore, chemotherapy and targeted therapy easily lead to drug resistance. Current technologies lack glycoprotein components with good specificity for women's cancers. To address this problem, this invention, after in-depth research, provides the following solution:

[0009] An anticancer active glycoprotein is a protein chemically bonded with selenium and polysaccharides, wherein the polysaccharide chain is a glycounit fragment having the structure of Formula 1.

[0010] Formula 1;

[0011] The proteins include peroxidase domain proteins, RNA-dependent RNA polymerase, the C-terminal domain protein of the nucleolar 278 pre-rRNA processing Urb / Npa2 (nucleolar pre-ribosome-associated protein 2), and ribosomal protein S12;

[0012] The hydroxyl groups in polysaccharides are linked to at least one of the following amino acids in proteins: aspartic acid, valine, glutamic acid, arginine, lysine, and serine, via hydrogen bonds.

[0013] Selenium bonds include at least one of Se=O and Se-CO.

[0014] This invention provides a novel selenoglycoprotein component, and further research shows that it can specifically inhibit female cancer cells and has low inhibitory activity against normal cells.

[0015] The polysaccharide portion of the anticancer active glycoprotein is composed of monosaccharide units of glucose, galactose, arabinose, and xylose linked by glycosidic bonds. In the hydrolyzed polysaccharide portion, the glucose content is above 25%, the galactose content is above 20%, the arabinose content is above 10%, and the contents of fucose, rhamnose, mannose, xylose, galacturonic acid, and glucuronic acid are all above 4%.

[0016] Preferably, the hydrolyzed heteropolysaccharide compound contains 28-30% glucose, 20-22% galactose, and 12-14% arabinose; and 4%-10% fucose, rhamnose, mannose, xylose, galacturonic acid, and glucuronic acid.

[0017] In this invention, the total content of the eight essential amino acids in the protein portion is 20-22 mg / g; among which, the alanine content is greater than 11 mg / g and the lysine content is greater than 5 mg / g.

[0018] The polysaccharide portion has a molecular weight of 10,000~11,000 Da; the protein portion has a molecular weight of 30~120 KDa.

[0019] Preferably, the selenium content in the anticancer active glycoprotein is 51~53 μg / g.

[0020] The present invention also provides a method for preparing the aforementioned anticancer active glycoprotein, wherein the mycelium powder of selenium-enriched Pleurotus pulmonale (also referred to as Pleurotus pulmonale mycelium in this invention) with accession number CCTCCNO: M 20242531 is defatted, salt extracted, and alcohol precipitated to obtain crude selenium glycoprotein (also marked as Se-POGP in this invention, or simply POGP);

[0021] The crude selenium glycoprotein was separated by a cellulose exchange column. The separation process included sequential elution with sodium chloride solution at a concentration of less than 0.15 mol / L and elution with sodium chloride solution at a concentration of 0.25~0.35 mol / L. The eluent from the 0.25~0.35 mol / L sodium chloride solution was collected as the target eluent and then desalted, concentrated, and purified by dextran gel column chromatography to obtain the anticancer active glycoprotein.

[0022] This invention innovatively involves defatting, salt extraction, and alcohol precipitation of the mycelium of selenium-enriched Pleurotus pulmonale (accession number CCTCC NO: M 20242531) without deproteinization to obtain crude selenium glycoprotein. Subsequently, by controlling the elution mechanism described above, a novel physicochemical structure can be obtained, which also exhibits excellent anticancer activity and selectivity against female cancer cells.

[0023] The preservation information of the selenium-enriched Pleurotus ostreatus described in this invention is as follows:

[0024] Strain: Selenium-enriched Pleurotus erythrorhizon (also known as Pleurotus erythrorhizon).

[0025] Preservation period: November 11, 2024;

[0026] China Center for Type Culture Collection (CCTCC);

[0027] Accession number: CCTCC NO: M 20242531;

[0028] Classified and named: Pleurotus pulmonae PpXN08 Pleurotus pulmonarius PpXN08;

[0029] Address: China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province.

[0030] In this invention, the solvent for the degreasing process is an aqueous alcohol solution, wherein the volume fraction of alcohol is 75-85%.

[0031] Preferably, the degreasing method is reflux degreasing.

[0032] Preferably, the defatted product is subjected to salt extraction in brine; wherein the brine used in the salt extraction process is a sodium chloride solution of 0.05~0.15 mol / L.

[0033] Preferably, the temperature for salt extraction is 85~95 ℃.

[0034] Preferably, alcohol is added to the extract after salt extraction for alcohol precipitation, followed by solid-liquid separation to obtain the alcohol precipitate. Additionally, during alcohol precipitation, the extract can be moderately concentrated as needed to save on alcohol usage. The amount of alcohol can be adjusted appropriately as required; for example, the volume ratio of extract (or its concentrate) to alcohol can be 1:1 to 10; more specifically, it can be 1:3 to 5.

[0035] Preferably, the alcohol precipitate is subjected to dialysis to obtain crude selenium glycoprotein.

[0036] In this invention, the cellulose exchange column is a DEAE-52 cellulose ion exchange column.

[0037] In this invention, during the elution process, water can be used for elution beforehand, followed by elution with a 0.1~0.15M sodium chloride solution, and then elution with the target eluent (0.25~0.35 mol / L sodium chloride solution), and the target eluent is collected.

[0038] Preferably, the target eluent is an eluent solution of sodium chloride solution with a concentration of 0.28~0.32 mol / L.

[0039] Preferably, the target eluent is desalted by dialysis.

[0040] Preferably, the dextran gel column is a G-100 dextran gel column.

[0041] The present invention also provides the application of the aforementioned anticancer active glycoprotein in the preparation of anti-female cancer drugs.

[0042] The application described in this invention is as an anticancer active ingredient used in a drug to inhibit female cancers caused by at least one cancer cell in Skov3, MDA-231, and HeLa cells.

[0043] The application described in this invention refers to female cancers including at least one of ovarian cancer, breast cancer, and cervical cancer.

[0044] The present invention also provides an anti-cancer drug for women, comprising a pharmaceutically effective amount of an anti-cancer active ingredient, wherein the anti-cancer active ingredient comprises the anti-cancer active glycoprotein described in the present invention.

[0045] The anti-cancer drug for women described in this invention also contains pharmaceutically acceptable excipients;

[0046] Preferably, it has a pharmaceutically acceptable dosage form.

[0047] Beneficial effects

[0048] 1. The present invention unexpectedly discovered that the active ingredient Se-POGP-2b has excellent anti-female cancer activity and selectivity.

[0049] 2. The present invention, through the preparation method described above, can obtain a Se-POGP-2b substance with good anti-cancer activity and no obvious toxic side effects on normal cells. Attached Figure Description

[0050] Figure 1 The molecular weight diagram of Se-POGP-2b is shown below.

[0051] Figure 2 Diagram showing the monosaccharide composition of Se-POGP-2b;

[0052] Figure 3 The cellulose elution curve of Se-POGP;

[0053] Figure 4 Elution curve of Se-POGP-2b;

[0054] Figure 5 The one-dimensional hydrogen NMR spectrum of Se-POGP-2b;

[0055] Figure 6 This is a one-dimensional carbon NMR spectrum of Se-POGP-2b;

[0056] Figure 7 Two-dimensional nuclear magnetic resonance of Se-POGP-2b 1 H- 1 H COSY diagram;

[0057] Figure 8 HSQC diagram for Se-POGP-2b;

[0058] Figure 9 HMBC diagram of Se-POGP-2b;

[0059] Figure 10 NOSEY plot of Se-POGP-2b;

[0060] Figure 11Here is the polysaccharide structure diagram of Se-POGP-2b;

[0061] Figure 12 The images show the protein structures identified by Se-POGP-2b; (a) is a diagram of ribosomal protein S12 (protein ID A0A8H7DQ59); (b) is a diagram of nucleolar proribosome-associated protein 2 (protein ID A0A067NRL7); (c) is a diagram of RNA-dependent RNA polymerase (protein ID A0A067NMT3); and (d) is a diagram of peroxidase domain protein (protein ID A0A067N671).

[0062] Figure 13 Here are the docking conformation diagrams of Se-POGP-2b; where (a) is the optimal conformation diagram of the docking of the peroxidase domain protein and Se-POGP-2b using Autodock software; and (b) is the optimal conformation diagram of the docking of nucleolar proribosome-associated protein 2 and Se-POGP-2b using Autodock software.

[0063] Figure 14 The diagram shows the docking conformation of Se-POGP-2b; where (a) is the optimal conformation diagram of ribosomal protein S12 and Se-POGP-2b docking using Autodock software; and (b) is the optimal conformation diagram of RNA-dependent RNA polymerase and Se-POGP-2b docking using Autodock software.

[0064] Figure 15 The figures show the CCK-8 experimental results for Se-POGP-2b; where a) is the result for Skov3, b) is the result for IOSE, c) is the result for MDA-231, d) is the result for MCF-10A, e) is the result for hela, and f) is the result for VK2.

[0065] Figure 16 Figures show the experimental results of CCK-8 for Se-POGP; where a) is the result for Skov3, b) is the result for IOSE, c) is the result for MDA-231, d) is the result for MCF-10A, e) is the result for hela, and f) is the result for VK2.

[0066] Figure 17 Figures show the results of AO / EB staining experiments of Se-POGP-2b on Skov3 and IOSE; where a) is the result of AO / EB staining experiment on Skov3 and b) is the result of AO / EB staining experiment on IOSE.

[0067] Figure 18Figures show the AO / EB staining results of Se-POGP-2b on MDA-231 and MCF-10A; where a) is the AO / EB staining result of MDA-231 and b) is the AO / EB staining result of MCF-10A.

[0068] Figure 19 Figures show the results of AO / EB staining experiments of Se-POGP-2b on Hela and VK2; where a) is the result of AO / EB staining experiment on Hela, and b) is the result of AO / EB staining experiment on VK2.

[0069] Figure 20 Figure showing the results of Se-POGP-2b cancer cell apoptosis experiments;

[0070] Figure 21 Graph showing changes in tumor growth in mice after administration of crude selenium glycoprotein (Se-POP) and Se-POGP-2b from Pleurotus pulmonarius mycelium;

[0071] Figure 22 Figure showing the changes in body weight gain in mice after administration of crude selenium glycoprotein (Se-POP) and Se-POGP-2b from Pleurotus pulmonarius mycelium;

[0072] Figure 23 Anatomical diagram of tumor size in mice after administration of crude selenium glycoprotein (Se-POP) and Se-POGP-2b from *Pleurotus pulmonarius* mycelium. Detailed Implementation

[0073] The following examples are intended to illustrate the invention and not to further limit it.

[0074] In this invention, mycelium is obtained by conventional culture of Pleurotus pulmonarius (CCTCC NO: M 20242531), such as liquid culture, and the mycelium is then used for subsequent extraction of active ingredients.

[0075] As an optional approach, the mycelium can be obtained by, for example, pre-culturing the *Pleurotus pulmonaryus* in a basal medium to obtain a mother culture, and then culturing the mother culture in a selenium-containing basal medium to obtain the mycelium of the *Pleurotus pulmonaryus* (also known as *Pleurotus pulmonaryus* mycelium).

[0076] The basal culture medium is an aqueous solution containing 150-250 g / L potato, 15-25 g / L glucose, 1-5 g / L yeast, 0.1-1 g / L magnesium sulfate and 0.5-1.5 g / L potassium dihydrogen phosphate.

[0077] In the selenium-containing basal culture medium, the selenium is sodium selenite. Its concentration can be 5~50 mg / L, and more specifically 10~20 mg / L.

[0078] The culture temperature can be 24~25℃, the stirring pressure can be controlled at 0.1~0.15 Pa, and the culture time can be more than 5 days, or even 5~15 days.

[0079] As an example, the mycelium used in the following cases was prepared through the following steps:

[0080] (1) Preparation of primary mother seeds

[0081] Pleurotus pulmonarius was inoculated into a liquid culture medium; after the mycelium had fully colonized the bags in the dark at 24°C in the culture room, the bags were opened at 24°C and humidity above 80% for fruiting management. After the fruiting bodies grew, the fruiting bodies were taken, and after tissue isolation and transfer, the primary mother culture was obtained.

[0082] Liquid culture medium formula: potato 200 g / L, glucose 20 g / L, yeast 3 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1 g / L;

[0083] (2) Preparation of liquid bacterial strains

[0084] The primary mother culture blocks were transferred to a selenium-containing liquid culture medium. 10-12 blocks with a diameter of 5 mm were inoculated and cultured on a shaker at 24℃ and 135 rpm / min for 8-10 days to obtain selenium-enriched liquid mycelium.

[0085] Selenium-containing liquid culture medium formula: potato 200 g / L, glucose 20 g / L, yeast 3 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1 g / L, sodium selenite concentration 15 mg / L;

[0086] (3) Liquid mycelium production

[0087] The primary mother culture liquid inoculum was inoculated into sterilized liquid culture medium containing selenium (15 mg / L). After culturing for 10 days at a temperature of 24-25℃ and a stirring pressure controlled at 0.1-0.15 Pa, the mycelial balls were filtered, rinsed three times with pure water, soaked and stirred for 15 min, filtered and dried to obtain mycelium.

[0088] Example 1

[0089] Extraction and separation experiments of Se-POGP-2b active ingredients:

[0090] (1) Defatting:

[0091] Weigh an appropriate amount of *Pleurotus pulmonarius* mycelium (preservation number CCTCC NO: M 20242531) powder, pour it into a round-bottom flask, add 80 v% ethanol solution (liquid-solid ratio of 5 mL / g), and reflux for defatting for 1 h.

[0092] (2) Salt extraction: Pour the defatted mycelium into a round-bottom flask, add 0.1M sodium chloride at a ratio of 1:20~25 (w / v, g / mL), and extract twice in a water bath at 85~95°C for 1.5 hours each time.

[0093] (3) Alcohol precipitation: The extract collected in (2) was concentrated (to 50% of the original volume), and then the concentrated extract was slowly poured into pre-cooled anhydrous ethanol at a ratio of 1:4, with constant stirring. After precipitation at 4°C overnight, the product was dialyzed, collected, and freeze-dried to obtain crude selenium glycoprotein (Se-POGP).

[0094] (4) Column chromatography purification: The crude selenium glycoprotein was purified by cellulose exchange column. The steps were as follows: 100 mg of lyophilized crude selenium glycoprotein was dissolved in 3 mL of water and slowly loaded along the edge. Gradient elution was performed with deionized water, 0.1, 0.3, 0.5, and 0.7 mol / L sodium chloride solutions. The sugar content was monitored by the phenol-sulfuric acid method (200 μL of eluent was taken, 100 μL of 0.5% phenol and 500 μL of concentrated sulfuric acid were added, mixed and cooled for 10 min, and the ultraviolet absorption at 490 nm was measured). The fraction eluted with 0.3 mol / L sodium chloride solution was collected, and the target eluent was dialyzed for desalting. Then, secondary purification was performed using a dextran gel column (the dextran gel column was a G-100 dextran gel column) to obtain the target active ingredient, which was named Se-POGP-2b active ingredient.

[0095] The selenium content in Se-POGP-2b is 52.1 ug / g.

[0096] The cellulose exchange column is a DEAE-52 cellulose ion exchange column.

[0097] Example 2

[0098] Se-POGP-2b (prepared in Example 1) methylation experiment:

[0099] Methylation: Weigh 3 mg of Se-POGP-2b sample into a glass reaction flask, add 1 mL of anhydrous DMSO, dissolve Se-POGP-2b in a suitable solvent in a reaction beaker, then add anhydrous alkaline solution A, sonicate to dissolve, and then add iodomethane solution B. Stir magnetically in a water bath at 30°C for 60 min, and finally add 2 mL of ultrapure water to terminate the methylation reaction.

[0100] Acid hydrolysis: The methylated polysaccharide was hydrolyzed in 1 mL of 2M trifluoroacetic acid (TFA) for 90 min, and then concentrated by rotary evaporation until no liquid remained.

[0101] Reduction: Add 2mL of double-distilled water and 60mg of sodium borohydride to reduce for 8 hours, then add glacial acetic acid to neutralize, rotary evaporate, and dry in an oven at 101 degrees Celsius.

[0102] Acetylation: Add 1 mL of acetic anhydride and react at 100 °C for 1 h, then cool. Add 3 mL of toluene, concentrate under reduced pressure and evaporate to dryness. Repeat 4-5 times to remove excess distilled water. After thorough shaking, remove the supernatant. Repeat this process 4 times. Dissolve the acetylated product in 3 mL of CH₂Cl₂, transfer to a separatory funnel, add a small amount of sodium sulfate, dry, concentrate to 1 mL, and transfer to a liquid chromatography vial. Analyze the acetylated product sample using a Thermo Scientific 1300-7000 gas chromatograph-mass spectrometer.

[0103] The mass spectra of the methylated sugar residues obtained in the experiment were compared with those of standard PMAA (partially methylated sugar alcohol acetyl esters), and the assignment and proportion of each sugar residue were determined based on retention time and mass-to-nucleus ratio. The analysis results of the GC-MS data are shown in Table 1.

[0104]

[0105] Example 3

[0106] Se-POGP-2b (prepared in Example 1) NMR experiment:

[0107] Take 50 mg of dried Se-POGP-2b sample, dissolve it in 500 μL of D2O, freeze-dry it three times, and then analyze the sample using nuclear magnetic resonance spectroscopy. The detection spectra include 1D NMR (…). 1 H NMR, 13 C NMR and 2D NMR (HH-COSY, HSQC, HMBC, NOESY).

[0108] Example 4

[0109] Determining the molecular structure of Se-POGP-2b (prepared in Example 1):

[0110] according to 1 H and 13 The anomeric region of the C-chromatic spectroscopy determines the number of anomeric hydrogens and carbons. Based on the chemical shifts of the anomeric hydrogens and carbons, the chemical shifts of other hydrogens associated with the anomeric proton are identified in the HH-COSY spectrum. Based on the proton's chemical shift, the chemical shifts of the carbons corresponding to the proton's chemical shift are identified in the HSQC spectrum. All sugar residues... 1 H, 13The C chemical shifts are shown in Table 2. After assigning the carbon and hydrogen chemical shifts, the carbon-hydrogen correlation signal peaks were identified using the HMBC spectrum to determine the linkage mode and sequence of sugar residues, thus completing the analysis of the NMR spectrum of the Se-POGP-2b component.

[0111]

[0112] Example 5

[0113] Se-POGP-2b protein identification and docking experiments:

[0114] In this invention, protein identification is mainly achieved by extracting proteins from samples, followed by reduction and alkylation, enzymatic digestion, desalting, and detection using LC-MS / MS.

[0115] After obtaining the protein structure, the available protein structures were searched in the database. Docking software was used to dock the polysaccharide with the protein to predict its optimal structure, and then visualization software such as PyMOL was used to visualize the structure. The results showed that the hydroxyl groups in the polysaccharide are connected to at least one amino acid in the protein, namely aspartic acid, valine, glutamic acid, arginine, lysine, and serine, through hydrogen bonds.

[0116] Example 6

[0117] Efficacy studies of Se-POGP-2b or Se-POGP (prepared in Example 1) against cancer cells (ovarian cancer, breast cancer, and cervical cancer) and normal cells (normal cells of the ovary, breast, and cervix):

[0118] Log-phase cancer cells (ovarian, breast, and cervical cancer) and normal cells (ovarian, breast, and cervical normal cells) were collected and digested with trypsin until the cells became rounded. Cell culture medium was then added to stop the digestion, and the cells were centrifuged at 1200 rpm for 5 min. The cell suspension was appropriately diluted, and cells were counted using a hemocytometer. 5 × 10⁶ cells were seeded per well in a 96-well plate. 3 Cells were cultured in a 37°C incubator for 24 hours.

[0119] Dissolve Se-POGP-2b or Se-POGP samples in basal culture medium to prepare Se-POGP-2b or Se-POGP solutions of different concentrations (0, 100, 200, 300, 400, 500, 600 μg / mL), and then sterilize by filtration through a 0.22 μm filter membrane.

[0120] Cell viability = (OD) 实验组 -OD 阴性对照组 ) / (OD 空白组 -OD 阴性对照组 )

[0121] To investigate the effects of Se-POGP-2b and Se-POGP on the growth status of cancer cells and normal cells, cancer cells and normal cells were prepared at a ratio of 1×10⁶ cells / mL in each confocal glass dish. 5 Cells were seeded at a density of 1000 μL and incubated at 37°C. After 24 hours, the old culture medium was discarded, and the cell surface was washed with PBS. 1 mL of fresh culture medium containing different concentrations of GDPs-1 was added to each dish. After another 24 hours of incubation, following the AO / EB kit instructions, 120 μL of a 10-fold diluted solution C, 15 μL of AO, and 15 μL of EB were added to each dish. The dishes were incubated at room temperature in the dark for 1-5 minutes, the dye was removed, the cells were washed once with PBS, and then soaked in 500 μL of PBS. Observation was performed using a confocal microscope (AO excitation 488 nm, emission 515 nm; EB excitation 518 nm, emission 605 nm).

[0122] To further demonstrate whether Se-POGP-2b and Se-POGP can induce cancer cell apoptosis, cells were collected and seeded into six-well plates at a density of 200,000 cells per well, and cultured at 37°C. After 24 hours, the cell surface was washed with PBS, and fresh medium containing different concentrations of Se-POGP-2b and Se-POGP was added to each well for further culture. After 24 hours, the old medium was discarded, and the cells were washed again with PBS. 500 μL of EDTA-free trypsin was added to each well for 1-5 min until the cells rounded. The cells were centrifuged, and the supernatant was discarded. 500 μL of prepared staining solution (500 μL 1×BB, 5 μL Annexin V-FITC, and 10 μL PI) was added to each tube, and the cells were resuspended. The cells were incubated at room temperature in the dark for 10 min, centrifuged, and the supernatant was discarded. 1 mL of PBS was added for resuspending. The cells were analyzed by flow cytometry within one hour using Flowjo software.

[0123] With increasing concentrations of Se-POGP-2b and Se-POGP, cancer cell growth was significantly inhibited in a concentration-dependent manner. For Skov3, MDA-231, and HeLa, the cell inhibition rates at a concentration of 600 μg / mL were 72% and 60%, 57% and 63%, and 62% and 27%, respectively. For IOSE, MCF-10A, and VK2, cell growth was not affected with increasing concentrations.

[0124] AO / EB staining results showed that when the Se-POGP-2b concentration was 0 μg / mL, cancer cells emitted uniform green fluorescence, and normal cell morphology with intact cell membranes could be observed in Blank. With increasing Se-POGP-2b concentration, the AO / EB fluorescence of cancer cells significantly increased, and cell membrane shrinkage and rounding were observed in Blank, indicating an increase in the number of apoptotic or necrotic cells, consistent with the results obtained from the previous CCK-8 experiment. The AO / EB staining results suggest that Se-POGP-2b and Se-POGP may mediate apoptosis in cancer cells, while having little effect on the growth of normal cells.

[0125] Apoptosis experiments showed that for cancer cells, with increasing Se-POGP-2b concentration, a significant increase in the proportion of cells in quadrants Q2 and Q3 of the apoptosis quadrant diagram was observed, while the proportion of cells in quadrant Q4 decreased, reaching 13.1% (Skov3), 50.9% (MDA-231), and 27.4% (HeLa) at the highest concentration, indicating a gradual increase in the number of apoptotic cells. Annexin V-FITC / PI and AO / EB staining results both confirmed that Se-POGP-2b can induce apoptosis in cancer cells without affecting the growth of normal cells.

[0126] Example 7

[0127] In vivo mouse animal experiments were conducted on the Se-POGP or Se-POGP-2b prepared in Example 1:

[0128] (1) Laboratory animals

[0129] Strain: Balb / c nude mouse (female, 4-6 weeks old, weight 16-18g);

[0130] Quantity: 56 (7 per group);

[0131] Keeping conditions: SPF-rated environment, 12-hour day-night cycle, free access to food and water;

[0132] (2) Grouping scheme (Table 3)

[0133]

[0134] (3) Establishment of tumor model

[0135] Cell line: Human ovarian cancer SKOV-3 cells (ATCC certified);

[0136] Seeding method: Logarithmic growth phase cells (5 × 10⁻⁶) 60.1 mL / mouse was injected subcutaneously into the ovarian tissue of mice.

[0137] Twelve hours after the last administration, all experimental mice were weighed and blood samples were taken. Subsequently, the mice were euthanized by cervical dislocation, and their spleens and ovaries (tumors) were carefully collected and accurately weighed.

[0138] Calculation formula: Organ index (mg / g) = Organ weight (mg) / Body weight (g);

[0139] Sample collection: Mice were sacrificed 12 hours after the last administration, and spleen and tumor tissue were separated under aseptic conditions.

[0140] Experimental results showed that, compared with the model group, the tumor weight in the drug group and the positive control group was smaller, indicating a better inhibitory effect. The spleen index can indicate the body's immune status and reflect the immunotoxicity of antitumor drugs; compared with the blank group, the spleen in the model group was significantly swollen. However, in the drug group, the spleen index was significantly lower than that in the model group, showing a dose-dependent effect. 5-FU treatment achieved a relatively high tumor inhibition rate, but compared with the model group, it also showed strong cytotoxic effects on these immune organs, which limits its further application.

Claims

1. An anticancer active glycoprotein, characterized in that, The protein is chemically bonded with selenium and polysaccharide, the polysaccharide chain is a sugar unit fragment with the structure of formula 1; Formula 1 The protein includes a peroxidase domain protein, an RNA-dependent RNA polymerase, a nucleolar 278 pre-rRNA processing Urb / Npa2 C-terminal domain protein, and a ribosomal protein S12; The hydroxyl in the polysaccharide is connected to at least one amino acid in aspartic acid, valine, glutamic acid, arginine, lysine, and serine in the protein through a hydrogen bond; The selenium bond includes at least one of Se=O and Se-C-O; The preparation method of the anticancer active glycoprotein is as follows: the mycelium powder of the selenium-rich Pleurotus eryngii is defatted, salted, and alcohol precipitated to obtain a crude selenium glycoprotein; The crude selenium glycoprotein is separated and treated by a cellulose exchange column, and the separation process includes sodium chloride solution elution processes with concentrations of 0.15 mol / L or less and 0.25-0.35 mol / L, which are sequentially performed; The eluate of the sodium chloride solution with a concentration of 0.25-0.35 mol / L is collected as a target eluate, and is desalted, concentrated, and purified by a dextran gel column to obtain the anticancer active glycoprotein.

2. The anticancer active glycoprotein of claim 1, wherein, In the protein part, the total content of eight essential amino acids in the human body is 20-22 mg / g; the content of alanine is greater than 11 mg / g, and the content of lysine is greater than 5 mg / g; The molecular weight of the polysaccharide part is 10,000-11,000 Da; and the molecular weight of the protein part is 30-120 KDa; The selenium content in the anticancer active glycoprotein is 51-53 μg / g.

3. A method for preparing the anticancer active glycoprotein according to claim 1 or 2, characterized by, The mycelium powder of the selenium-rich Pleurotus eryngii with the preservation number CCTCC NO: M 20242531 is defatted, salted, and alcohol precipitated to obtain a crude selenium glycoprotein; The crude selenium glycoprotein is separated and treated by a cellulose exchange column, and the separation process includes sodium chloride solution elution processes with concentrations of 0.15 mol / L or less and 0.25-0.35 mol / L, which are sequentially performed; The eluate of the sodium chloride solution with a concentration of 0.25-0.35 mol / L is collected as a target eluate, and is desalted, concentrated, and purified by a dextran gel column to obtain the anticancer active glycoprotein.

4. The method of preparing an anticancer active glycoprotein according to claim 3, wherein the step of preparing the anti-cancer active glycoprotein is performed by the method according to any one of claims 1 to 3. The solvent for the defatting process is an alcohol aqueous solution, wherein the volume fraction of the alcohol is 75-85%; The defatting method is reflux defatting; The defatted product is salted in brine; wherein the brine for the salting process is a sodium chloride solution with a concentration of 0.05-0.15 mol / L; The salting temperature is 85-95 ℃; Alcohol is added to the extracted solution after salting to perform alcohol precipitation treatment, and then solid-liquid separation is performed to obtain an alcohol precipitate; The alcohol precipitate is dialyzed to obtain the crude selenium glycoprotein.

5. The method of preparing an anticancer active glycoprotein according to claim 4, wherein the step of preparing the anti-cancer active glycoprotein is performed by the method of claim 1. The cellulose exchange column is a DEAE-52 cellulose ion exchange column; Dialysis is used to desalt the target eluate; The dextran gel column is a G-100 dextran gel column.

6. Use of an anticancer active ingredient for the preparation of a medicament against cancer in a female, characterized in that, The anticancer active ingredient includes the anticancer active glycoprotein according to any one of claims 1 to 2 and / or the anticancer active glycoprotein prepared by the preparation method according to any one of claims 3 to 5. The female cancer is at least one of ovarian cancer, breast cancer, and cervical cancer.

7. Use according to claim 6, wherein The anticancer active glycoprotein is used as an anticancer active ingredient for preparing a medicine for inhibiting female cancer caused by at least one of Skov3, MDA-231, and Hela cells.

8. An anti-cancer drug for women, comprising a pharmaceutically effective amount of an anticancer active ingredient, characterized in that, The anticancer active ingredient includes the anticancer active glycoprotein according to any one of claims 1 to 2 or the anticancer active glycoprotein prepared by the preparation method according to any one of claims 3 to 5.

9. The anti-female cancer drug according to claim 8, wherein It further includes a pharmaceutically acceptable adjuvant; It has a pharmaceutically acceptable administration form.

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