A grifola frondosa glycoprotein extract, its preparation method and use

Abstract

The application belongs to the technical field of natural product extraction and medicine, and discloses a grifola frondosa glycoprotein extract, a preparation method and application thereof.The grifola frondosa glycoprotein extract GFP60-1 has a molecular weight of about 2 kDa, a purity higher than 90%, and a monosaccharide composition containing only glucose; the grifola frondosa glycoprotein extract contains 17 kinds of amino acids such as lysine, arginine, histidine, alanine and threonine; the grifola frondosa glycoprotein extract is obtained by using grifola frondosa fruiting bodies as raw materials, and through hot water extraction, ethanol fractional precipitation and anion exchange processes; and the grifola frondosa glycoprotein extract has an anti-radiation protection effect.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and pharmaceutical technology, specifically relating to a glycoprotein extract extracted from the fruiting body of Grifola frondosa, its preparation method, and its application in the preparation of anti-radiation drugs. Background Technology

[0002] With the development of fields such as nuclear energy, medical radiation, and aerospace, the risk of human exposure to ionizing radiation (IR) continues to increase. Gamma rays are typically produced by the decay of radioactive nuclides (such as... 60 Co、 137 The radiation produced by ionizing radiation (Cs) is high in energy and has strong penetrating power, which can cause systemic radiation damage. Ionizing radiation can directly cause DNA double-strand breaks and induce the radiation decomposition of intracellular water molecules to produce a large amount of reactive oxygen species (ROS), which in turn triggers oxidative stress reactions such as lipid peroxidation and protein damage, leading to acute radiation sickness, immune system suppression, hematopoietic function damage and intestinal flora imbalance, which can be life-threatening in severe cases.

[0003] Currently, the most commonly used radiation protectant in clinical practice is amifostine (WR-2721), an aminothiol compound, which is the only approved radiation protectant for clinical use to date. However, amifostine has a short half-life, inconvenient administration routes, and significant toxic side effects such as reversible hypotension, nausea, and vomiting, severely limiting its clinical application. There is an urgent need to develop novel, safe, and highly effective radiation protectants. Natural polysaccharides, as an important class of bioactive substances, have advantages such as high safety and wide availability, and their radiation protective activity has received widespread attention in recent years.

[0004] Grifola frondosa, also known as Maitake, is a fungus used for both food and medicinal purposes. It contains various active ingredients such as polysaccharides and their complexes, proteins, and glycopeptides. To date, research on the active ingredients of Grifola frondosa has mainly focused on polysaccharides and their complexes, which possess various biological activities including antioxidant, immunomodulatory, and antitumor effects. Some studies have also preliminarily demonstrated its radioprotective properties. In recent years, reports on the activity and function of Grifola frondosa glycoproteins and glycopeptides have gradually increased. For example, patent CN114230646A discloses an antitumor Grifola frondosa glycoprotein, its preparation method, and its application. This glycoprotein has a protein content of 54.0±3.0%, a polysaccharide content of 20.3±2.0%, a weight-average molecular weight of 17.5 kDa, and contains seven monosaccharides including fucose, glucosamine hydrochloride, galactose, and glucose, as well as eleven amino acids including alanine, glycine, and threonine. However, previous studies have failed to elucidate the specific molecular mechanisms by which Grifola frondosa polysaccharides and their complexes exert radioprotective effects, and there is a lack of systematic isolation, structural characterization, and activity evaluation of Grifola frondosa low-molecular-weight glycoprotein components. Further research by researchers is urgently needed to enhance the medicinal value of the various active ingredients in Grifola frondosa. Summary of the Invention

[0005] In view of the shortcomings and deficiencies of the existing technology, the primary objective of this invention is to provide a Grifola frondosa glycoprotein extract GFP60-1, whose structural characteristics have been systematically characterized, and which has a defined molecular weight, monosaccharide and amino acid composition.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned Grifola frondosa glycoprotein extract. The preparation method of the present invention uses Grifola frondosa fruiting bodies as raw materials and employs processes such as hot water extraction, ethanol fractionation precipitation, and anion exchange to easily extract, separate, and purify a high-purity Grifola frondosa glycoprotein extract with a purity higher than 90%.

[0007] A third objective of this invention is to provide the application of the above-mentioned Grifola frondosa glycoprotein extract in the preparation of anti-radiation drugs. The inventors have conducted in-depth animal studies to investigate its anti-radiation activity and molecular mechanism, providing a theoretical basis and technical support for the development of naturally derived radiation protectants.

[0008] The objective of this invention is achieved through the following technical solution: A Grifola frondosa glycoprotein extract, wherein the extract has a molecular weight of 1.5~2.5 kDa and a purity of not less than 90%; the monosaccharide composition contains only glucose, and the amino acid composition includes 17 kinds of amino acids: lysine, arginine, histidine, alanine, threonine, serine, valine, glycine, aspartic acid, proline, glutamic acid, isoleucine, leucine, tyrosine, methionine, phenylalanine and cysteine.

[0009] Preferably, the amino acids contain 18-22% lysine, 17-21% arginine, 10-14% histidine, 8-12% alanine, and 7-11% threonine, respectively; among which, the essential amino acids include threonine, valine, methionine, isoleucine, leucine, phenylalanine, and lysine, accounting for more than 38%.

[0010] Preferably, the extract has a zeta potential of -2.0 to -4.0 mV and an average particle size of less than 500 nm.

[0011] The preparation method of the above-mentioned Grifola frondosa glycoprotein extract includes the following steps: (1) Dry and grind the fruiting bodies of Grifola frondosa into powder and sieve; defatt the powder with ethanol, centrifuge and air dry to obtain defatted powder; (2) Take defatted powder, add water to extract, centrifuge to obtain supernatant, concentrate; then remove free protein, rotary evaporate to remove organic solvent, and obtain concentrated solution; (3) Add ethanol to the concentrate, let stand, centrifuge and take the supernatant; add ethanol to the supernatant, let stand overnight, centrifuge and collect the precipitate, collect the precipitate fraction, add water to redissolve, remove alcohol by rotary evaporation, concentrate and freeze dry to obtain Grifola frondosa crude polysaccharide GFP60. (4) GFP60 was prepared into an aqueous solution, and then subjected to anion exchange column chromatography with NaCl aqueous solution gradient elution. The main elution peak component was collected, dialyzed to desalt, and freeze-dried to obtain Grifola frondosa glycoprotein extract GFP60-1.

[0012] The Grifola frondosa glycoprotein extract of this invention can be prepared by extraction, separation and purification processes such as hot water extraction, ethanol fractionation precipitation and anion exchange. The operation is simple, the purity is high and the extract has high activity.

[0013] As a preferred option, in step (1), the product is passed through a 40-80 mesh sieve, and the degreasing time is 16-24 h at room temperature.

[0014] Preferably, in step (2), the ratio of material to liquid for extraction is 1:15 to 1:25 g / mL, the temperature is 60 to 80℃, and the time is 4 to 6 h.

[0015] Preferably, in step (3), the ethanol concentration in the system is 40-60% after adding ethanol to the concentrate, and the ethanol concentration in the system is 60-80% after adding ethanol to the supernatant.

[0016] Preferably, in steps (1), (2), and (3), the centrifugation conditions are 8000 r / min, the temperature is 4℃, and the time is 5~10 min.

[0017] Preferably, in step (4), the concentration of the prepared GFP60 aqueous solution is 1~5 mg / mL, and the concentration of the NaCl aqueous solution is 0.05~0.5 mol / L.

[0018] The above-mentioned application of Grifola frondosa glycoprotein extract in the preparation of anti-radiation drugs. Grifola frondosa glycoprotein extract GFP60-1 has anti-γ-ionizing radiation function, which is manifested in improving serum IgA, IgM and IgG levels; improving spleen and thymus organ indices; reducing mouse serum MDA levels and increasing SOD and GSH-Px activities; and protecting the normal function of the hematopoietic system, etc. Therefore, it can be used to prepare anti-radiation drugs.

[0019] Therefore, the present invention has the following beneficial effects: (1) The present invention successfully extracted Grifola frondosa glycoprotein extract GFP60-1, with a molecular weight of about 2kDa, containing only glucose monosaccharides and 17 kinds of amino acids. The extraction method is simple to operate. (2) This invention confirms that Grifola frondosa glycoprotein extract GFP60-1 has anti-γ-ionizing radiation function and can be used to prepare anti-radiation drugs, as shown in the following: In animal experiments, Grifola frondosa glycoprotein extract GFP60-1 can improve the effect of 5 Gy of radiation. 60 Co-γ irradiation of mice showed the following effects: increased spleen and thymus organ indices; increased serum IgA, IgM, and IgG levels; decreased serum MDA levels and increased SOD and GSH-Px activities; alleviated damage to erythrocytes, leukocytes, and platelets, while effectively protecting DNA in bone marrow, thus protecting the normal function of the hematopoietic system; improved intestinal and spleen damage; improved radiation-induced intestinal flora imbalance, effectively increased the Chao1 and Shannon indices in the mouse intestinal flora, improved α and β diversity of the intestinal flora in irradiated mice, and increased the relative abundance of beneficial bacteria such as Lachnospiraceae_NK4A136_group and Alistipes. Attached Figure Description

[0020] Figure 1 The molecular weight chromatogram of GFP60-1, a glycoprotein extract from Grifola frondosa.

[0021] Figure 2 The image shows the monosaccharide composition of GFP60-1, a glycoprotein extract from Grifola frondosa.

[0022] Figure 3 The image shows the UV absorption spectrum of GFP60-1, a glycoprotein extract from Grifola frondosa.

[0023] Figure 4 The FT-IR infrared spectrum of GFP60-1, a glycoprotein extract from Grifola frondosa.

[0024] Figure 5 Glycopeptide bond characterization diagram of GFP60-1, a glycoprotein extract from Grifola frondosa.

[0025] Figure 6 This is a particle size distribution diagram of GFP60-1, a glycoprotein extract from Grifola frondosa.

[0026] Figure 7 The effect of Grifola frondosa glycoprotein extract GFP60-1 on mouse body weight.

[0027] Figure 8 The effect of Grifola frondosa glycoprotein extract GFP60-1 on organ indices in mice; in the figure, (A) spleen index; (B) thymus index.

[0028] Figure 9 The effect of Grifola frondosa glycoprotein extract GFP60-1 on the immunoglobulin content in mouse serum; in the figure, (A) IgA level; (B) IgM level; (C) IgG level.

[0029] Figure 10 The effect of Grifola frondosa glycoprotein extract GFP60-1 on MDA, SOD and GSH-Px in mouse serum; in the figure, (A) MDA; (B) SOD; (C) GSH-Px.

[0030] Figure 11 The effect of Grifola frondosa glycoprotein extract GFP60-1 on DNA damage in mouse bone marrow.

[0031] Figure 12 The effect of Grifola frondosa glycoprotein extract GFP60-1 on intestinal damage in mice.

[0032] Figure 13 The effect of Grifola frondosa glycoprotein extract GFP60-1 on spleen damage in mice.

[0033] Figure 14 The effect of Grifola frondosa glycoprotein extract GFP60-1 on ASV in mouse gut microbiota; in the figure, (A) the number of OTUs for each sample; (B) the Venn diagram based on ASV.

[0034] Figure 15 The effect of Grifola frondosa glycoprotein extract GFP60-1 on the Alpha diversity of mouse gut microbiota; in the figure, (A) Chao1 index; (B) Shannon index; (C) Simpson index.

[0035] Figure 16 For sequencing curve analysis; in the figure, (A) dilution curve; (B) aroma index curve; (C) species accumulation curve; (D) abundance level curve.

[0036] Figure 17 The effect of Grifola frondosa glycoprotein extract GFP60-1 on the Beta diversity of mouse gut microbiota; in the figure, (A) PCoA; (B) NMDS; (C) UPGMA.

[0037] Figure 18 The effects of Grifola frondosa glycoprotein extract GFP60-1 on the phylum and genus levels of mouse gut microbiota are shown in the figure. (A) Phylogenetic abundance map; (B) Phylogenetic heatmap; (C) Genus abundance map; (D) Genus heatmap; (EI) Significance analysis of microbiota abundance.

[0038] Figure 19 LEfSe analysis of different groups of mouse gut microbiota; in the figure, (A) LDA value distribution bar chart; (B) evolutionary branching diagram. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments of the present invention are not limited thereto.

[0040] Example 1

[0041] The specific steps for preparing Grifola frondosa glycoprotein extract are as follows: (1) Raw material pretreatment: Dry and grind the fruiting bodies of Grifola frondosa into powder, and pass them through a 40-mesh sieve; add 95% ethanol to defatt the powder at room temperature for 16 hours, centrifuge and air dry to obtain defatted powder; (2) Hot water extraction: Take defatted powder, add deionized water at a material-to-liquid ratio of 1:15 g / mL, and extract in hot water at 60℃ for 4h; then centrifuge at 8000 r / min and 4℃ for 5 min to collect the supernatant, and rotary evaporate under reduced pressure at 80 r / min, 40℃ and -0.08 MPa; remove free protein by Sevag method, remove organic solvent by rotary evaporation to obtain concentrated solution; (3) Ethanol fractionation and precipitation: Add ethanol to the concentrate until the final ethanol concentration in the system is 40%. After standing, centrifuge at 8000 r / min and 4℃ for 5 min and discard the precipitate. Take the supernatant. Continue to add ethanol to the supernatant until the final ethanol concentration in the system is 60%. After standing overnight, centrifuge at 8000 r / min and 4℃ for 5 min, collect the precipitate, redissolve in water, remove alcohol by rotary evaporation, concentrate and freeze dry to obtain Grifola frondosa crude polysaccharide GFP60. (4) Anion exchange column chromatography: GFP60 was prepared into an aqueous solution with a concentration of 1 mg / mL, loaded onto a DEAE Sepharose Fast Flow column, and eluted with a gradient of 0.05 mol / L NaCl aqueous solution. The main peak component was collected by eluting with deionized water and then freeze-dried to obtain Grifola frondosa glycoprotein extract GFP60-1.

[0042] The molecular weight, purity, composition, particle size and zeta potential of the Grifola frondosa glycoprotein extract GFP60-1 obtained in Example 1 were determined, and infrared spectroscopy and ultraviolet spectroscopy were performed.

[0043] (1) Determination of molecular weight The molecular weight of GFP60-1, a glycoprotein extract from Grifola frondosa, was determined by high-performance gel permeation chromatography (HPLC) using a Shimadzu LC2030 HPLC system with a RID-20 differential detector and TSKgel G3000PWXL and TSKgel G4000PWXL gel permeation columns. The sample was dissolved in deionized water to prepare an aqueous solution with a concentration of 2 mg / mL. A standard curve was established using dextran standards with molecular weights of 500, 1000, 3620, 7000, 9000, 20000, 50000, 126000, 250000, 490000, 1000000, and 2000000 Da for calibration. Results are shown below. Figure 1 As shown in the chromatogram, the single peak of Grifola frondosa glycoprotein extract GFP60-1 accounted for a high proportion, with a purity of 90.38%, and the peak shape was round and narrow, indicating that DEAE Sepharose Fast Flow anion exchange chromatography had a good purification effect on Grifola frondosa glycoprotein extract GFP60-1. Based on the standard curve and the elution time of Grifola frondosa glycoprotein extract GFP60-1 (38.65 min), the weight-average molecular weight of Grifola frondosa glycoprotein extract GFP60-1 in Example 1 can be determined to be approximately 2 kDa.

[0044] (2) Monosaccharide composition analysis The monosaccharide composition of GFP60-1, a glycoprotein extract from Grifola frondosa, was determined using a combination of PMP pre-column derivatization high-performance liquid chromatography and chemical methods. The specific operational steps are as follows: Derivatization of sample a: Prepare a 5 mg / mL solution of Grifola frondosa glycoprotein extract GFP60-1. Add 100 µL of each solution to an EP tube, add 3 mL of 2.0 mol / L trifluoroacetic acid (TFA) solution, and react at 110℃ for 5 h. After the reaction, cool and add 3 mL of methanol, remove TFA by rotary evaporation, add methanol several times to completely remove TFA; then add 1 mL of distilled water and sonicate to dissolve. b. Derivatization of monosaccharide standards: Prepare a 5 mg / mL monosaccharide standard solution. Mix 200 µL of each monosaccharide standard with 100 µL of standard solution, add 1000 µL of 0.5 mol / L PMP methanol solution and 100 µL of 0.3 mol / L sodium hydroxide solution, and hydrolyze in a water bath at 70 °C for 1 h. After hydrolysis, cool to room temperature, add 100 µL of 0.3 mol / L hydrochloric acid, and extract with 1.0 mL of chloroform. Centrifuge at 12000 r / min for 2 min. c. Liquid chromatography conditions: Acetonitrile and phosphate buffer (volume ratio 18:82) were used as the mobile phase, C18 column was used for separation, flow rate was 0.5 mL / min, detection wavelength was 245 nm, injection volume was 20 µL, and run time was 90 min.

[0045] The results are as follows Figure 2 As shown, in Example 1, only glucose was found as a monosaccharide in the Grifola frondosa glycoprotein extract GFP60-1.

[0046] (3) Amino acid composition analysis Five mg of Grifola frondosa glycoprotein extract GFP60-1 was dissolved in 5 mL of 6 mol / L hydrochloric acid, filled with nitrogen gas, and sealed in a vacuum tube. The solution was hydrolyzed at 110 °C for 22 h. The resulting mixture was neutralized with sodium hydroxide solution and then filtered through a 0.22 μm filter membrane. The hydrolysis products were analyzed using an automated amino acid analyzer, and the results are shown in Table 1.

[0047] Table 1. Amino acid composition of Grifola frondosa glycoprotein extract GFP60-1

[0048] Note: * indicates essential amino acids.

[0049] Analysis revealed that the Grifola frondosa glycoprotein extract GFP60-1 obtained in this invention contains 17 amino acids, with the main amino acids being lysine, arginine, histidine, alanine, and threonine, accounting for 20.33%, 19.38%, 12.03%, 10.22%, and 9.78%, respectively. It also contains significant proportions of valine, glycine, aspartic acid, proline, glutamic acid, isoleucine, and leucine. Furthermore, the proportion of essential amino acids reached 38.09%.

[0050] (4) Ultraviolet spectroscopy analysis A certain amount of Grifola frondosa glycoprotein extract GFP60-1 was weighed and dissolved in deionized water to prepare a 5 mg / mL glycoprotein extract solution. The sample was then scanned in the 200–400 nm range using a UV spectrophotometer (GENESYS 150). The results are as follows: Figure 3 As shown, it exhibits an absorption peak at 260-280 nm, indicating the presence of certain proteins and amino acids.

[0051] (5) FT-IR infrared spectroscopy analysis Two mg of dried Grifola frondosa glycoprotein extract GFP60-1 was mixed with 100–200 mg of KBr powder, ground in a mortar, and compressed into tablets. The tablets were then analyzed using a Fourier transform infrared spectrophotometer at 4000 cm⁻¹. -1 Up to 400 cm -1 Spectral analysis was performed within the specified range. The results are as follows: Figure 4 As shown, the Grifola frondosa glycoprotein extract GFP60-1 exhibits distinct polysaccharide characteristics at 3329 cm⁻¹. -1 The stretching vibration absorption peak of -OH was observed at 1631 cm⁻¹. -1 and 1414 cm -1 The absorption peak at that location may be due to the asymmetric and symmetric stretching of the carboxylic acid anionic group (C=O), indicating the presence of a protein.

[0052] (6) Glycopeptide bond analysis Take 1 mL of the prepared 2 mg / mL Grifola frondosa glycoprotein extract GFP60-1 aqueous solution, add 1 mL of 0.4 mol / L NaOH solution, and place in a 45℃ water bath for 2 h. After cooling to 25℃, perform UV scanning in the wavelength range of 200-400 nm. Compare the changes before and after the reaction to determine the type of glycopeptide bond. The results are as follows: Figure 5 As shown, the absorption peak of the Grifola frondosa glycoprotein extract GFP60-1 at around 240 nm changed after NaOH treatment. This indicates that after NaOH treatment, the monosaccharide or polysaccharide chains linked to the serine or threonine hydroxyl groups on the peptide chain were hydrolyzed, leading to a β-elimination reaction of the O-glycopeptide bonds. The corresponding serine and threonine were converted into α-aminopropionic acid and α-aminobutenoic acid, thereby enhancing the absorption at 240 nm. This demonstrates that the extract GFP60-1 of this invention is a glycoprotein rather than a polysaccharide, and the protein and polysaccharide of GFP60-1 are linked by O-type glycopeptide bonds.

[0053] (7) Determination of particle size and zeta potential Five mg of Grifola frondosa glycoprotein extract GFP60-1 was dissolved in 5 mL of deionized water and thoroughly mixed. The particle size and zeta potential were determined using a laser particle size analyzer at 25°C. The results are as follows: Figure 6 As shown, its average particle size is less than 500 nm, which falls within the particle size range of nanopolysaccharides. The Zeta potential of Grifola frondosa glycoprotein extract GFP60-1 is approximately -3.032 mV, indicating that its particle surface carries a negative charge.

[0054] Example 2

[0055] The specific steps for preparing Grifola frondosa glycoprotein extract are as follows: (1) Raw material pretreatment: Dry and grind the fruiting bodies of Grifola frondosa into powder, and pass them through a 40-mesh sieve; add 95% ethanol to defatt the powder at room temperature for 20 hours, centrifuge and air dry to obtain defatted powder; (2) Hot water extraction: Take defatted powder, add deionized water at a material-to-liquid ratio of 1:20 g / mL, and extract in hot water at 70℃ for 5h; then centrifuge at 8000 r / min and 4℃ for 8 min to collect the supernatant, and rotary evaporate under reduced pressure at 80 r / min, 40℃ and -0.08 MPa; remove free protein by Sevag method, remove organic solvent by rotary evaporation to obtain concentrated solution; (3) Ethanol fractionation and precipitation: Add ethanol to the concentrate until the final ethanol concentration in the system is 40%. After standing, centrifuge at 8000 r / min and 4℃ for 8 min and discard the precipitate. Take the supernatant. Continue to add ethanol to the supernatant until the final ethanol concentration in the system is 80%. After standing overnight, centrifuge at 8000 r / min and 4℃ for 8 min, collect the precipitate, redissolve in water, remove alcohol by rotary evaporation, concentrate and freeze dry to obtain Grifola frondosa crude polysaccharide GFP60. (4) Anion exchange column chromatography: GFP60 was prepared into an aqueous solution with a concentration of 2 mg / mL, loaded onto a DEAE Sepharose Fast Flow column, and eluted with a gradient of 0.2 mol / L NaCl aqueous solution. The main peak component was collected by eluting with deionized water and then freeze-dried to obtain Grifola frondosa glycoprotein extract GFP60-1.

[0056] Example 3

[0057] The specific steps for preparing Grifola frondosa glycoprotein extract are as follows: (1) Raw material pretreatment: Dry and grind the fruiting bodies of Grifola frondosa into powder, and pass them through a 40-mesh sieve; add 95% ethanol to defatt the powder at room temperature for 24 hours, centrifuge and air dry to obtain defatted powder; (2) Hot water extraction: Take defatted powder, add deionized water at a material-to-liquid ratio of 1:25 g / mL, and extract in hot water at 80℃ for 6h; then centrifuge at 8000 r / min and 4℃ for 10 min to collect the supernatant, and rotary evaporate under reduced pressure at 80 r / min, 40℃ and -0.08 MPa; remove free protein by Sevag method, remove organic solvent by rotary evaporation to obtain concentrated solution; (3) Ethanol fractionation and precipitation: Add ethanol to the concentrate until the final ethanol concentration in the system is 60%. After standing, centrifuge at 8000 r / min and 4℃ for 10 min and discard the precipitate. Take the supernatant. Continue to add ethanol to the supernatant until the final ethanol concentration in the system is 80%. After standing overnight, centrifuge at 8000 r / min and 4℃ for 10 min, collect the precipitate, redissolve in water, remove alcohol by rotary evaporation, concentrate and freeze dry to obtain Grifola frondosa crude polysaccharide GFP60. (4) Anion exchange column chromatography: GFP60 was prepared into an aqueous solution with a concentration of 5 mg / mL, loaded onto a DEAE Sepharose Fast Flow column, and eluted with a gradient of 0.5 mol / L NaCl aqueous solution. The main peak component was collected by eluting with deionized water and then freeze-dried to obtain Grifola frondosa glycoprotein extract GFP60-1.

[0058] Examples 2 and 3 adjusted the ethanol defatting time, extraction liquid-to-solid ratio, and other conditions, and successfully prepared Grifola frondosa glycoprotein extract.

[0059] Application examples To determine the anti-radiation effect of the Grifola frondosa glycoprotein extract GFP60-1 of the present invention, an in vivo anti-radiation study was conducted on mice using the Grifola frondosa glycoprotein extract obtained in Example 1.

[0060] I. Laboratory Animals The experimental animals were 6-week-old male SPF-grade BALB / c mice (weighing 20 ± 2 g), purchased from the Experimental Animal Center of Hangzhou Medical College. The mice were housed in a pathogen-free barrier environment with a constant temperature of 22 ± 2℃, relative humidity of 60% ± 5%, and alternating light / dark cycles of 12 h each. They were fed standard mouse diet (AIN-93) and sterile water. SPF-grade animal usage guidelines were strictly followed to ensure the health and safety of the experimental animals.

[0061] II. Experimental Methods After 7 days of acclimatization, the mice were randomly divided into 7 groups of 10 mice each. The grouping details are as follows: Normal blank control group (NC): Free access to sterile water and feed daily for 21 days; Radiation damage model group (MC): Same as the blank control group, with free access to sterile water and feed for 21 days; Positive drug control group (PC): mice were given free access to sterile water daily and were given an intraperitoneal injection of amifostine 0.3 mL per mouse 1 hour before irradiation, with a total dose of 150 mg / kg. Low-dose group of Grifola frondosa glycoprotein extract (GFPL): Grifola frondosa glycoprotein extract GFP60-1 solution was administered by gavage at the same time every day for 21 days at a dose of 100 mg / kg·bwt. Medium-dose group of Grifola frondosa glycoprotein extract (GFPM): Grifola frondosa glycoprotein extract GFP60-1 solution was administered by gavage at the same time every day for 21 days at a dose of 200 mg / kg·bwt. High-dose group of Grifola frondosa glycoprotein extract (GFPH): Grifola frondosa glycoprotein extract GFP60-1 solution was administered by gavage at the same time every day for 21 days at a dose of 400 mg / kg·bwt.

[0062] Except for the NC group mice, the other mice received 5 Gy of irradiation at the Zhejiang Provincial Irradiation Center on day 22. 60 Co-γ radiation was administered via whole-body irradiation. No mice died during the entire administration process, and no abnormalities were observed in appearance or behavior.

[0063] III. Data Processing Statistical analysis was performed using IBM SPSS Statistics 20 software. ANOVA was used for analysis of variance. Charts were generated using Origin 9.0 and GraphPad Prism 9 software. Each experiment was performed in at least three replicates. Data are presented as mean plus standard deviation (SD).

[0064] IV. Experimental Results (1) Weight measurement The mice were weighed and recorded on the first day before gavage, before irradiation treatment, and before sacrifice and dissection. The results are as follows: Figure 7 As shown, all groups of mice showed significant weight gain after 21 days of feeding, but the weight gain of mice in the Grifola frondosa glycoprotein extract GFP60-1 group was not significant. Compared to the NC group, the MC group mice showed a significant weight loss after irradiation, indicating that the radiation damage model was successfully established. Compared to the MC and PC groups, the weight loss of mice in each dose group of Grifola frondosa glycoprotein extract was not significant after irradiation, indicating that Grifola frondosa glycoprotein extract GFP60-1 reduced the trend of weight loss in mice to some extent.

[0065] (2) Determination of organ index During the dissection of mice, the thymus and spleen were weighed, and the thymus index and spleen index were calculated. The results are as follows: Figure 8As shown, after mice were pretreated with Grifola frondosa glycoprotein extract GFP60-1, their immune organ indices increased to varying degrees, and this preventive effect showed a dose-dependent relationship with Grifola frondosa glycoprotein extract, indicating that GFP60-1 has a certain regulatory effect on the immune system of mice after radiation.

[0066] (3) Measurement of peripheral blood counts in mice The collected whole blood from mice was immediately placed in anticoagulant EP tubes, and the number of white blood cells (WBC), red blood cells (RBC), and platelets (PLT) in the peripheral blood of each group of mice was detected using a blood analyzer. The results are shown in Table 2. Different lowercase letters in the same column indicate significant differences between the data (p<0.05).

[0067] Table 2 Peripheral blood counts in mice

[0068] Compared to the NC group, the MC group showed a significant decrease in white blood cell, red blood cell, and platelet counts, indicating that ionizing radiation causes significant damage to these cells, especially white blood cells. Comparing the peripheral blood counts of mice in the MC group and the various dose groups of Grifola frondosa glycoprotein extract, the white blood cell, red blood cell, and platelet counts were increased to some extent in all dose groups compared to the MC group. This suggests that the intake of Grifola frondosa glycoprotein extract GFP60-1 can effectively prevent this damage. The groups administered medium (200 mg / kg) and high (400 mg / kg) doses of Grifola frondosa glycoprotein showed better radiation protection in mice, with red blood cell counts even exceeding those in the positive control group (PC). Therefore, this indicates that Grifola frondosa glycoprotein extract GFP60-1 has a certain protective effect on the blood system of mice exposed to ionizing radiation.

[0069] (4) Determination of immunoglobulins Following the manufacturer's instructions, the levels of immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) in the serum of mice in each group were measured using an ELISA kit. The results are as follows: Figure 9 As shown, radiation caused some damage to the immune system of mice. The levels of IgA, IgM and IgG in the serum of mice in the radiation damage model group decreased to varying degrees. However, the serum levels of IgA, IgM and IgG in mice treated with each dose of Grifola frondosa glycoprotein extract GFP60-1 were higher than those in the model group, especially in IgM and IgG. This indicates that Grifola frondosa glycoprotein extract has a protective effect on the immune system of mice exposed to ionizing radiation.

[0070] (5) Determination of MDA, SOD and GSH-Px in serum Following the manufacturer's instructions, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) levels in the serum of mice in each group were measured to assess the balance between the body's oxidation and antioxidant systems.

[0071] MDA is the end product of lipid peroxidation, and its level directly reflects the severity of oxidative damage in the body. Results are as follows... Figure 10 As shown in (A), although the ability of Grifola frondosa glycoprotein extract GFP60-1 to reduce MDA is not as good as that of amifostine, the pretreatment with Grifola frondosa glycoprotein extract significantly reduced the MDA level in mouse serum compared with the radiation damage model group mice. High-dose treatment with Grifola frondosa glycoprotein extract reduced the MDA concentration in mouse serum to 8.03 ± 0.60 nmol / mL.

[0072] Superoxide dismutase (SOD) is the first line of defense in antioxidant protection, catalyzing superoxide anion radicals into hydrogen peroxide. The results are as follows... Figure 10 As shown in (B), radiation significantly inhibited SOD activity in mouse serum. High-dose Grifola frondosa glycoprotein extract played the most significant role in restoring SOD activity in mouse serum after irradiation. In addition, the low- and medium-dose Grifola frondosa glycoprotein extract groups also had a certain restorative effect on SOD activity compared with the radiation damage model group.

[0073] GSH-Px primarily scavenge lipid peroxides and reduce cell membrane damage. Results are as follows: Figure 10 As shown in (C), 5 Gy of γ radiation caused a decrease in GSH-Px activity in mouse serum. Different doses of Grifola frondosa glycoprotein extract GFP60-1 all had the effect of restoring GSH-Px activity, but the low and medium doses of Grifola frondosa glycoprotein extract did not show significant effects. However, the high dose group of Grifola frondosa glycoprotein extract had a significant preventive and protective effect on GSH-Px activity in mouse serum after irradiation treatment.

[0074] In summary, high-dose Grifola frondosa glycoprotein extract can reduce serum MDA levels and increase serum SOD and GSH-Px activities in irradiated mice, indicating that Grifola frondosa glycoprotein extract has an anti-radiation effect.

[0075] (6) Bone marrow DNA damage assay The left femur of the mouse was dissected. After removing the muscle tissue, both ends of the femur were cut open. The bone marrow was washed into a centrifuge tube with 10 mL of 0.005 mol / L CaCl2 solution. After multiple washes, the tube was incubated at 4°C for 30 min. The tube was then centrifuged at 2500 r / min for 15 min. The precipitate was collected and acidified with 5 mL of 0.2 mol / L HClO4. After thorough mixing, the tube was placed in a 90°C water bath for 15 min. After cooling with running water, the tube was centrifuged at 2500 r / min for 15 min. The supernatant was collected, and the absorbance of the filtrate was measured at 260 nm using a UV-Vis spectrophotometer to calculate the DNA content in the bone marrow. The results are as follows: Figure 11 As shown, compared with the NC group, the bone marrow DNA content of mice in the radiation damage model group was significantly reduced. However, the bone marrow DNA of mice in each dose group of Grifola frondosa glycoprotein extract and the positive drug control group remained at a high level. In particular, the high dose (400 mg / kg) of Grifola frondosa glycoprotein extract could significantly prevent this damage, which indicates that Grifola frondosa glycoprotein extract has the potential to protect against radiation-induced hematopoietic damage.

[0076] (7) Observation of organizational morphology After euthanizing the mice, the collected colon and spleen tissues were fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. Then, 4 µm sections were cut, stained with hematoxylin and eosin (H&E), and used for histological observation.

[0077] Results of mouse intestinal tissue Figure 12 As shown, the colonic pathological morphology of mice in the NC and PC groups was normal, with intact colonic mucosa and clearly visible mucosal crypt structures. The thickness of the crypt base was normal, and the number of goblet cells was relatively large, indicating relatively intact intestinal function. In the MC group, a large number of neutrophils were aggregated in the colon, showing abnormal inflammatory infiltration. Furthermore, the crypt structures were blurred, and the intestinal mucosa was damaged, indicating that radiation caused significant damage to the intestinal tissue of the MC group mice. Mice in the GFPL group still showed some inflammatory infiltration in their intestinal tissue, but this was alleviated compared to the MC group, and the crypt structures were clearer, indicating that low-dose Grifola frondosa glycoprotein extract GFP60-1 had a certain protective effect against intestinal damage after radiation in mice. In the medium- and high-dose Grifola frondosa glycoprotein extract GFP60-1 groups, the colonic crypt structures were clear, the number of goblet cells was similar to that in the normal and PC groups, there was no obvious abnormal inflammatory infiltration, and the mucosa was intact, indicating that medium- and high-dose Grifola frondosa glycoprotein extract GFP60-1 could reduce the impact of radiation on the mouse colon. Therefore, the Grifola frondosa glycoprotein extract GFP60-1 has a certain preventive effect on radiation-induced intestinal damage in mice.

[0078] Results of mouse spleen tissue examination: Figure 13 As shown, the spleen tissue morphology in the NC group was normal, with abundant lymphocytes in the periarterial lymphatic sheath of the spleen (black arrows), and no obvious histopathological changes were observed. In contrast, the spleen of mice in the radiation damage model group showed diffuse necrosis (yellow arrows) and lysis, with a significant decrease and disappearance of lymphocytes, indicating severe spleen damage. The spleen damage in the PC and GFPL groups was somewhat alleviated, but regional necrosis and numerous apoptotic bodies remained (within black circles). The diffuse necrosis in the spleen of mice in the GFPM group was further reduced, with a significant decrease in apoptotic bodies, and the spleen tissue morphology tended to be normal. The spleen morphology of mice in the GFPH group was closest to normal, with fewer apoptotic bodies and an increased number of lymphocytes. The above comparisons demonstrate that the Grifola frondosa glycoprotein extract GFP60-1 is significantly effective in preventing radiation-induced spleen damage.

[0079] (8) Intestinal flora analysis DNA was extracted from mouse fecal sediment, and the 16S rDNA gene in the V3-V4 region was amplified by PCR. Custom barcode primers 341F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') were used. Samples were assembled using FLASH software to obtain tag data. Alpha diversity of the samples was analyzed using Mothur software, including dilution curves, aroma index curves, and abundance ranking curves. Alpha and beta diversity analyses were performed using QIIME software. Principal component analysis (PCA) and principal coordinate analysis (MDS) were performed using R language and the Binary Jaccard method. Taxonomic analysis was performed by comparing OTUs with databases. Finally, species abundance analysis was performed using QIIME software to generate community structure maps.

[0080] The effects of Grifola frondosa glycoprotein extract GFP60-1 on mouse gut microbiota amplicon sequence variants (ASVs) are as follows: Figure 14 As shown, a total of 2164 ASVs were identified in the sequence, including 452 in the NC group, 397 in the MC group, 401 in the PC group, and 416, 520, and 490 in the GFPH, GFPM, and GFPL groups, respectively, with a total of 322 ASVs in all groups. Compared with the NC group, the number of ASVs in the MC group decreased after irradiation. However, after gavage administration of different doses of Grifola frondosa glycoprotein extract GFP60-1, the number of ASVs in the mice increased, indicating that Grifola frondosa glycoprotein extract GFP60-1 can alleviate radiation-induced dysbiosis.

[0081] The effects of Grifola frondosa glycoprotein extract GFP60-1 on Alpha diversity of mouse gut microbiota are as follows: Figure 15 As shown, radiation caused a decrease in the Shannon index and an increase in the Simpson index in the gut microbiota of mice, while the Grifola frondosa glycoprotein extract GFP60-1 increased the diversity of the gut microbiota in mice after irradiation to a certain extent and alleviated radiation-induced dysbiosis.

[0082] Sequencing curves are used to assess whether the sequencing depth has reached the target; the results are as follows: Figure 16 As shown in (A) and (B), the dilution curve and the aroma index curve tend to plateau as the number of sequenced species increases, indicating that species diversity has reached saturation and the sequencing volume is sufficient. Figure 16 (C) shows that with a current sample size of 24, the species accumulation curve tends to flatten, indicating that the sequencing library is large enough that even with further increases in sequencing volume, the species level is saturated, covering most of the bacterial diversity in the sample. This also indicates that the sampling is sufficient and the data is reliable. The rank abundance curve, plotted after ranking species abundance, is used to assess the richness and evenness of the sample; the curve width reflects richness—the wider the curve, the richer the species composition; the curve trend reflects evenness—the flatter the curve, the more even the species composition. Figure 16 (D) shows that there are differences in richness and evenness among the groups, indicating that the sequencing depth is sufficient to cover most species.

[0083] The effects of Grifola frondosa glycoprotein extract GFP60-1 on the beta diversity of mouse gut microbiota are as follows: Figure 17 As shown, in Figure 17 (A) In PCoA, most of the NC group samples were located in the lower left corner, while the MC group samples were mostly far from the NC group, located in the lower right corner. This indicates that the gut microbiota composition of the MC group mice changed after radiation exposure. Mice in the different dose groups of Grifola frondosa glycoprotein extract showed a pattern closer to the NC group in PCoA, suggesting that gavage administration of Grifola frondosa glycoprotein extract GFP60-1 improved the gut microbiota of the mice. Further analysis... Figure 17 Analysis of NMDS in (B) yielded results similar to those of PCoA. (By...) Figure 17 (C) UPGMA analysis revealed a significant difference between the NC and MC groups, indicating that radiation had a significant modeling effect on the MC group mice, and the intestinal flora of the MC group mice underwent significant changes. In contrast, the GFPL group was close to the MC group, suggesting that low-dose Grifola frondosa glycoprotein extract GFP60-1 had no significant effect on improving the intestinal flora of mice, while medium and high doses of Grifola frondosa glycoprotein extract GFP60-1 had a significant effect on improving the intestinal flora of mice. The mice in both groups were closest to the NC group in branching. This indicates that after gavage administration of medium and high doses of Grifola frondosa glycoprotein extract GFP60-1, the composition of the intestinal flora in mice was restored.

[0084] The effects of Grifola frondosa glycoprotein extract GFP60-1 on the phylum and genus levels of mouse gut microbiota are as follows: Figure 18 As shown. By Figure 18 (A, B, E, and F) show that the relative abundance of Bacteroidetes in the gut microbiota of MC group mice increased compared to NC group, while the relative abundance of Firmicutes decreased. Intake of Grifola frondosa glycoprotein extract GFP60-1 caused the relative abundance of Bacteroidetes and Firmicutes in the gut microbiota of mice after irradiation to approach that of the NC group. Figure 18 (C, D, G, H, and I) indicate that radiation caused an increase in the relative abundance of Bacteroides and a decrease in the relative abundance of Lachnospiraceae_NK4A136_group in the mouse gut microbiota, and the intake of Grifola frondosa glycoprotein extract GFP60-1 effectively improved this situation; while Alistipes, as a common beneficial bacterium in the gut, had a significantly reduced relative abundance due to radiation, but Grifola frondosa glycoprotein extract GFP60-1 could increase its relative abundance in the gut.

[0085] The results of the differential analysis of mouse gut microbiota among different groups using Grifola frondosa glycoprotein extract GFP60-1 are as follows: Figure 19 As shown, when the LDA threshold is 3.5, 7, 8, 1, 4, 9, and 1 species in the NC, MC, PC, GFPL, GFPM, and GFPH groups, respectively, exhibited significant differences. At the class level, Campylobacteria in the NC group, Desulfovibrionia in the MC group, Saccharimonadia in the GFPM group, and Alphaproteobacteria in the GFPL group were the most significantly different species compared to other groups. At the order level, Campylobacterales in the NC group, Desulfovibrionales in the MC group, Clostridia_UCG_014, Clostridia_vadinBB60_group, Saccharimonadales in the GFPM group, and Rhodospirillales in the GFPL group were the most significantly different species compared to other groups. Furthermore, at the family level, Tannerellaceae and Desulfovibrionaceae in the MC group and Saccharimonadaceae in the GFPM group were the most significantly different species compared to other groups. The aforementioned visualization LEfSe analysis showed that, compared to the NC group, radiation produced certain inter-group differences in the gut microbiota structure of mice. Compared to the MC group, the microbial composition of mice in the GFPL and GFPH groups was reduced.

[0086] The above measurements verified the anti-radiation properties of Grifola frondosa glycoprotein extract GFP60-1, which are mainly reflected in the following aspects: (1) increasing the serum IgA, IgM and IgG levels in irradiated mice and improving the organ indices of the spleen and thymus; (2) alleviating oxidative stress damage induced by ionizing radiation, including reducing the level of lipid peroxidation product MDA in mice and increasing the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px); (3) reducing the damage of γ radiation to peripheral blood erythrocytes, leukocytes and platelets, protecting the hematopoietic system of mice, and reducing bone marrow DNA damage; (4) effectively increasing the Chao1 and Shannon indices in the intestinal flora of mice, improving the α and β diversity of the intestinal flora of irradiated mice, and increasing the relative abundance of beneficial bacteria such as Lachnospiraceae_NK4A136_group and Alistipes. Therefore, Grifola frondosa glycoprotein extract GFP60-1 can be used to prepare anti-radiation drugs.

Claims

1. A Grifola frondosa glycoprotein extract, characterized in that, The extract has a molecular weight of 1.5~2.5 kDa and a purity of not less than 90%; its monosaccharide composition contains only glucose, and its amino acid composition includes 17 kinds of amino acids: lysine, arginine, histidine, alanine, threonine, serine, valine, glycine, aspartic acid, proline, glutamic acid, isoleucine, leucine, tyrosine, methionine, phenylalanine and cysteine.

2. The Grifola frondosa glycoprotein extract according to claim 1, characterized in that, The amino acids contain 18-22% lysine, 17-21% arginine, 10-14% histidine, 8-12% alanine, and 7-11% threonine, respectively; among them, essential amino acids include threonine, valine, methionine, isoleucine, leucine, phenylalanine, and lysine, accounting for more than 38%.

3. The Grifola frondosa glycoprotein extract according to claim 1 or 2, characterized in that, The extract has a zeta potential of -2.0 to -4.0 mV and an average particle size of less than 500 nm.

4. A method for preparing the Grifola frondosa glycoprotein extract as described in any one of claims 1 to 3, characterized in that, step as follows: (1) Dry and grind the fruiting bodies of Grifola frondosa into powder, sieve, defatt with ethanol, centrifuge and air dry to obtain defatted powder; (2) Take defatted powder, add water to extract, centrifuge to obtain supernatant, concentrate; then remove free protein, rotary evaporate to remove organic solvent, and obtain concentrated solution; (3) Add ethanol to the concentrate, let stand, centrifuge and take the supernatant; add ethanol to the supernatant, let stand overnight, centrifuge, collect the precipitate, add water to redissolve, remove alcohol by rotary evaporation, concentrate and freeze dry to obtain Grifola frondosa crude polysaccharide GFP60. (4) GFP60 was prepared into an aqueous solution, and then subjected to anion exchange column chromatography with NaCl aqueous solution gradient elution. The main elution peak component was collected, dialyzed to desalt, and freeze-dried to obtain Grifola frondosa glycoprotein extract GFP60-1.

5. The preparation method according to claim 4, characterized in that, In step (1), the product is passed through a 40-80 mesh sieve and degreased at room temperature for 16-24 hours.

6. The preparation method according to claim 4, characterized in that, In step (2), the ratio of material to liquid for extraction is 1:15~1:25 g / mL, the temperature is 60~80℃, and the time is 4~6 h.

7. The preparation method according to claim 4, characterized in that, In step (3), after adding ethanol to the concentrate, the ethanol concentration in the system is 40-60%, and after adding ethanol to the supernatant, the ethanol concentration in the system is 60-80%.

8. The preparation method according to claim 4 or 7, characterized in that, In step (4), the concentration of the prepared GFP60 aqueous solution is 1~5 mg / mL, and the concentration of the NaCl aqueous solution is 0.05~0.5 mol / L.

9. The preparation method according to claim 4, characterized in that, In steps (1), (2) and (3), the centrifugation conditions are 8000 r / min, 4℃, and 5~10 min.

10. The use of the Grifola frondosa glycoprotein extract as described in any one of claims 1 to 3 or the Grifola frondosa glycoprotein extract prepared by any one of claims 4 to 9 in the preparation of anti-radiation drugs.

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