Polysaccharide compound having clear molecular structure and suitable for treating pulmonary fibrosis

Abstract

The present invention is applicable to the technical field of plant extraction and separation. Provided is a polysaccharide compound GLP-4 that has a clear molecular structure and has the effects of treating emphysema and pulmonary fibrosis and resisting tumors, which polysaccharide compound has a molecular formula of: (C162H270O135)n. Ganoderma is subjected to high-temperature and high-pressure extraction using an alkali solution, the resulting medicine residues are extracted, and then separation is performed by column chromatography to obtain the polysaccharide compound. The polysaccharide compound has a good water solubility, is easily absorbed by a human body, has better effects than pirfenidone in terms of the treatment of emphysema and pulmonary fibrosis, and also has efficacy in patients with advanced cancers (patients who have lost surgical condition, have a survival time of only three to six months, and still have chemotherapy condition). Especially when being used in combination with a chemotherapeutic drug, the polysaccharide compound can eliminate the toxic side effects caused by the chemotherapeutic drug in the human body, and can be used for controlling and reducing a tumor mass, and reducing and eliminating tumor cells, which effects are all supported by corresponding experimental data.

Description

A polysaccharide compound with a clear molecular structure suitable for the treatment of pulmonary fibrosis Technical Field

[0001] The present invention belongs to the field of plant extraction and separation technology improvement, and in particular relates to a method for extracting Ganoderma lucidum polysaccharide GLP-4 and its application. Background Art

[0002] Ganoderma lucidum is a fungal plant with a long history of medicinal use in China and Japan. The active ingredients in Ganoderma lucidum are numerous and complex in composition. Over 150 compounds have been isolated from it, primarily including polysaccharides, triterpenes, sterols, alkaloids, furan derivatives, aminopeptides, and inorganic elements. Different geographic locations (longitude and latitude), different seeds, different growing environments, and varying temperatures, humidity, and light levels can significantly affect the content, proportion, and presence of the active ingredients described in this article in Ganoderma lucidum.

[0003] Summary of the Invention

[0004] The purpose of the present invention is to provide a polysaccharide compound with a clear molecular structure and the effects of treating emphysema, pulmonary fibrosis and anti-tumor.

[0005] The present invention aims to address the currently unsolved treatment challenges for emphysema and pulmonary fibrosis. Currently, there are no effective treatments for these conditions. The emergence of GLP-4, a polysaccharide compound from Ganoderma lucidum, promises to fill this gap. GLP-4 also demonstrates excellent efficacy in treating advanced cancers, particularly when used in combination with chemotherapy drugs. It also prevents, controls, and treats the transformation of pulmonary fibrosis and lung nodules into lung cancer.

[0006] Another important function of the medicinal ingredients referred to in the present invention is to prevent the mutation of normal human cells and the generation of cancer cells.

[0007] The present invention is achieved by providing a method for extracting GLP-4 from Ganoderma lucidum polysaccharide, the method comprising the following steps:

[0008] S1, removing dust from the Ganoderma lucidum, drying it, and then crushing it to prepare Ganoderma lucidum powder;

[0009] S2, placing the crushed Ganoderma lucidum in a sealed container, mixing with water and heating, and fully blending the Ganoderma lucidum powder and water into a medicinal juice solution under high temperature and high pressure;

[0010] S3, using centrifugal technology to separate the medicinal juice solution to obtain a concentrated solution with medicinal components and medicinal residues;

[0011] S4, mixing the separated medicinal residues with NaOH solution according to a preset ratio, soaking them, and then adding HCl for neutralization;

[0012] S5. Concentrating and desalting the mixed solution by membrane concentration technology to remove NaCl components in the mixed solution to obtain a concentrated solution containing the medicinal components;

[0013] S6. The concentrated solution containing the medicinal ingredients is freeze-dried and prepared into a certain concentration, and then separated by column chromatography for multiple times to obtain Ganoderma lucidum polysaccharide GLP-4 containing the medicinal ingredients.

[0014] A further technical solution of the present invention is: in step S2, the Ganoderma lucidum powder and water in the sealed container are mixed and stirred thoroughly, and then heated to 105-200° C., and the boiling time is continued for 2-6 hours. As the heating temperature increases, the pressure inside the sealed container gradually increases to form a high-temperature and high-pressure environment in the sealed container.

[0015] A further technical solution of the present invention is: in step S2, the mixed liquid of Ganoderma lucidum powder and water in the sealed container is heated to 105-170° C., and the boiling time is continued for 3-6 hours. As the heating temperature increases, the pressure inside the sealed container gradually increases to form a high-temperature and high-pressure environment in the sealed container.

[0016] A further technical solution of the present invention is: in step S6, the concentrated lyophilized powder containing the medicinal ingredients is added with pure water to be prepared into 40-80 mg / ml, and is separated by multiple column chromatography to obtain the concentrate.

[0017] A further technical solution of the present invention is: in step S4, the medicinal residues are mixed and soaked with NaOH solution at a ratio of 1:10-1:40 at a temperature of 40-100° C. for 1-5 hours, wherein the concentration of NaOH is 0.05-0.5 mol / L.

[0018] A further technical solution of the present invention is: in step S3, the extract is separated from the Ganoderma lucidum residue by centrifugation technology to obtain the Ganoderma lucidum residue, which is then further extracted and purified.

[0019] A further technical solution of the present invention is: in step S1, the Ganoderma lucidum is washed with clean water to remove dust on the surface and then dried at 105° C. The dried Ganoderma lucidum is crushed, and the crushed Ganoderma lucidum powder is larger than 60 mesh.

[0020] A further technical solution of the present invention is: in the step S2, the mixed liquid in the closed container is heated to 105°C, 110°C, 115°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, 170°C, 175°C, 180°C, 185°C, 190°C, 195°C or 200°C, and the boiling time is 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h, and the pressure inside the closed container gradually increases as the heating temperature increases, forming a high temperature and high pressure environment of the closed container.

[0021] Another object of the present invention is to provide a Ganoderma lucidum polysaccharide GLP-4, the structural formula of which is Molecular formula (C 162 H 270 O 135 ) n , where n = 10-15.

[0022] A further technical solution of the present invention is: n is 10, 11, 12, 13, 14 or 15.

[0023] Another object of the present invention is to provide an application of GLP-4, a polysaccharide of Ganoderma lucidum. The polysaccharide GLP-4 of Ganoderma lucidum has good water solubility, is easily absorbed by the human body, and has therapeutic effects on emphysema, pulmonary fibrosis and tumors. In particular, the therapeutic effect on emphysema and pulmonary fibrosis is extremely excellent, significantly better than pirfenidone. When used in combination with chemotherapy drugs, it can eliminate the toxic side effects of chemotherapy drugs on the human body and has a certain effect on controlling and reducing tumor masses and reducing and eliminating tumor cells.

[0024] The invention has the following advantages: the extraction process is simple, the residue of Ganoderma lucidum is fully utilized, the production cost is low, the polysaccharide extraction rate is high, and the operation is simple. Ganoderma lucidum polysaccharide has good solubility, extremely low (or even no) toxic side effects, and is easily absorbed and degraded by the human body, thereby exerting its therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG1 is a flow chart of a method for extracting GLP-4, a polysaccharide of Ganoderma lucidum, provided in an embodiment of the present invention.

[0026] FIG2 is a schematic diagram of a 1gMp-RT (peak molecular weight) calibration curve provided in an embodiment of the present invention.

[0027] FIG3 is a schematic diagram of a 1gMp-RT (weight average molecular weight) calibration curve provided in an embodiment of the present invention.

[0028] FIG4 is a schematic diagram of a 1gMp-RT (number average molecular weight) calibration curve provided in an embodiment of the present invention.

[0029] FIG5 is a schematic diagram of the molecular weight spectrum of Ganoderma lucidum polysaccharide GLP-4 provided in an embodiment of the present invention.

[0030] FIG6 is a schematic diagram of an ion chromatogram of a mixed standard of 16 sugars provided in an embodiment of the present invention.

[0031] FIG7 is a second schematic diagram of an ion chromatogram of a mixed standard of 16 sugars provided in an embodiment of the present invention.

[0032] FIG8 is a GCMS chromatogram of Ganoderma lucidum polysaccharide GLP-4 (PMAA) provided in an embodiment of the present invention.

[0033] FIG9 is a schematic diagram 1 of the result analysis of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0034] FIG10 is a second schematic diagram of the analysis results of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0035] FIG11 is a third schematic diagram of the analysis results of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0036] FIG12 is a fourth schematic diagram of the analysis results of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0037] FIG13 is a fifth schematic diagram of the analysis results of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0038] FIG14 is a sixth schematic diagram of the analysis results of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0039] FIG15 is a schematic diagram of a hydrogen spectrum provided by an embodiment of the present invention.

[0040] FIG16 is a schematic diagram of a carbon spectrum provided by an embodiment of the present invention.

[0041] FIG17 is a schematic diagram of a Dept135 spectrum provided in an embodiment of the present invention.

[0042] FIG18 is a schematic diagram of HH-COSY provided by an embodiment of the present invention.

[0043] FIG19 is a HSQC diagram provided by an embodiment of the present invention.

[0044] FIG20 is an HMBC spectrum provided by an embodiment of the present invention.

[0045] FIG21 is a schematic diagram of NOESY provided by an embodiment of the present invention.

[0046] FIG22 is a schematic diagram showing the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the tumor volume of Lewis cell tumor-bearing mice provided by an embodiment of the present invention.

[0047] Figure 23 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on tumor pathology in Lewis cell tumor-bearing mice (×200) provided by an embodiment of the present invention.

[0048] Figure 24 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on renal pathology in Lewis cell tumor-bearing mice (×200) provided by an embodiment of the present invention.

[0049] Figure 25 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on gastric pathology in Lewis cell tumor-bearing mice (×200) provided by an embodiment of the present invention.

[0050] Figure 26 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on spleen pathology in Lewis cell tumor-bearing mice (×200) provided by an embodiment of the present invention.

[0051] Figure 27 is a schematic diagram of the control group of the H22 liver cancer-bearing mouse model treated with Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy provided in an embodiment of the present invention.

[0052] Figure 28 is a schematic diagram of the Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy in the cisplatin group of H22 liver cancer-bearing mice provided by an embodiment of the present invention.

[0053] Figure 29 is a schematic diagram of the cisplatin + GLP-4 low-dose group of H22 liver cancer-bearing mice treated with Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy provided by an embodiment of the present invention.

[0054] Figure 30 is a schematic diagram of the Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy for the cisplatin + GLP-4 high-dose group of H22 liver cancer-bearing mice provided by an embodiment of the present invention.

[0055] Figure 31 is a schematic diagram of the control group of the H22 liver cancer-bearing mouse tumor model treated with Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy provided in an embodiment of the present invention.

[0056] Figure 32 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the cisplatin group of H22 liver cancer-bearing mice provided by an embodiment of the present invention.

[0057] Figure 33 is a schematic diagram of the cisplatin + GLP-4 low-dose group for the treatment of H22 liver cancer-bearing mice treated with Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy provided by an embodiment of the present invention.

[0058] Figure 34 is a schematic diagram of the cisplatin + GLP-4 high-dose group for the treatment of H22 liver cancer-bearing mice treated with GLP-4 combined with chemotherapy according to an embodiment of the present invention.

[0059] Figure 35 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 orthotopic liver cancer tumors in mice (binning: 8×8; T=30s) provided by an embodiment of the present invention.

[0060] Figure 36 is a schematic diagram showing the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 orthotopic liver cancer tumors in mice provided by an embodiment of the present invention.

[0061] Figure 37 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the liver of mice with H22 in situ liver cancer (×200) provided by an embodiment of the present invention.

[0062] Figure 38 is a schematic diagram (×200) showing the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the spleen of mice with H22 orthotopic liver cancer provided by an embodiment of the present invention.

[0063] Figure 39 is a schematic diagram (×200) showing the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the stomach of mice with H22 in situ liver cancer provided by an embodiment of the present invention.

[0064] Figure 40 is a schematic diagram (×200) showing the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the kidneys of mice with H22 in situ liver cancer provided by an embodiment of the present invention.

[0065] FIG41 is a confocal image of a blank control group of RAW264.7 macrophages provided in an embodiment of the present invention under a 40x microscope.

[0066] Figure 42 is a confocal image at 40 times magnification after 0.25 mg / mL Ganoderma lucidum polysaccharide GLP-4 provided in an embodiment of the present invention was incubated with RAW264.7 macrophages for 24 hours.

[0067] Figure 43 is a confocal image under a 63x oil microscope after incubating 0.25 mg / mL Ganoderma lucidum polysaccharide GLP-4 with RAW264.7 macrophages for 24 hours provided by an embodiment of the present invention.

[0068] Figure 44 is a confocal image at 40 times magnification after 0.5 mg / mL Ganoderma lucidum polysaccharide GLP-4 provided in an embodiment of the present invention was incubated with RAW264.7 macrophages for 24 hours.

[0069] Figure 45 is a confocal image under a 63x oil microscope after incubating 0.5 mg / mL Ganoderma lucidum polysaccharide GLP-4 with RAW264.7 macrophages for 24 hours provided by an embodiment of the present invention.

[0070] Figure 46 is a schematic diagram showing the effects of the combination of Ganoderma lucidum polysaccharide GLP-4 and cisplatin on the necrosis rate and apoptosis rate of H22 cells provided by an embodiment of the present invention.

[0071] Figure 47 is a schematic diagram of the control group of the 4T1 breast cancer tumor-bearing mouse model treated with GLP-4, a Ganoderma lucidum polysaccharide, combined with chemotherapy, according to an embodiment of the present invention.

[0072] Figure 48 is a schematic diagram of the Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy in the cisplatin group of 4T1 breast cancer-bearing mice provided by an embodiment of the present invention.

[0073] Figure 49 is a schematic diagram of the Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy for the cisplatin + GLP-4 low-dose group of 4T1 breast cancer-bearing mice provided by an embodiment of the present invention.

[0074] Figure 50 is a schematic diagram of the Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy for the cisplatin + GLP-4 high-dose group of 4T1 breast cancer-bearing mice provided by an embodiment of the present invention.

[0075] Figure 51 is a schematic diagram of the control group of the 4T1 breast cancer tumor-bearing mouse model treated with Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy provided in an embodiment of the present invention.

[0076] Figure 52 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the cisplatin group of 4T1 breast cancer-bearing mice provided by an embodiment of the present invention.

[0077] Figure 53 is a schematic diagram of the cisplatin + GLP-4 low-dose group for the treatment of 4T1 breast cancer-bearing mice treated with Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy provided by an embodiment of the present invention.

[0078] Figure 54 is a schematic diagram of the cisplatin + GLP-4 high-dose group for the treatment of 4T1 breast cancer-bearing mice treated with Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy provided by an embodiment of the present invention.

[0079] FIG55 is a schematic diagram of lung tissue pathology (×40) of normal group mice provided in an embodiment of the present invention.

[0080] FIG56 is a schematic diagram of the lung tissue pathology (×40) of mice in the model control group provided in an embodiment of the present invention.

[0081] FIG57 is a schematic diagram of the lung tissue pathology (×40) of mice in the positive control group provided in an embodiment of the present invention.

[0082] Figure 58 is a schematic diagram of the lung tissue pathology (×40) of mice in the low-dose group of Ganoderma lucidum polysaccharide GLP-4 provided in an embodiment of the present invention.

[0083] Figure 59 is a schematic diagram of the lung tissue pathology (×40) of mice in the high-dose group of Ganoderma lucidum polysaccharide GLP-4 provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0084] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0085] As shown in FIG1 , the flow chart of the method for extracting GLP-4 from Ganoderma lucidum polysaccharide provided by the present invention is described in detail as follows:

[0086] Step S1, cleaning the harvested Ganoderma lucidum or Ganoderma lucidum that has undergone a preliminary simple treatment with clean water on a cleaning device, removing dust from the surface thereof, transferring the Ganoderma lucidum to a drying device for drying at a drying temperature of 105° C., placing the dried Ganoderma lucidum on a crusher for crushing into Ganoderma lucidum powder, sieving the Ganoderma lucidum powder, transferring the Ganoderma lucidum powder particles larger than 60 mesh to the next process, and returning the Ganoderma lucidum powder particles smaller than 60 mesh to the crushing mechanism for re-crushing, repeating the process multiple times until the crushed Ganoderma lucidum powder particles meet the specified requirements.

[0087] Step S2: mixing the qualified Ganoderma lucidum powder with pure water and placing the mixture in a sealed container, heating the sealed container at a high temperature. During the heating process, the pressure inside the sealed container gradually increases as the temperature rises, so that a high temperature and high pressure environment is formed in the sealed container. In this environment, it is easier to fully fuse the Ganoderma lucidum powder and water to form a mixed liquid. The sealed container is heated to a temperature between 105° C. and 200° C. for a boiling time of 2 to 6 hours, preferably between 105° C. and 170° C. for a boiling time of 3 to 6 hours. 6h, it is more preferably high temperature heating to 105℃, 110℃, 115℃, 120℃, 125℃, 130℃, 135℃, 140℃, 145℃, 150℃, 155℃, 160℃, 165℃, 170℃, 175℃, 180℃, 185℃, 190℃, 195℃ or 200℃, with a boiling time of 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h, so that the mixed solution is fully fused to form an aqueous solution containing the medicinal ingredients.

[0088] Step S3, using centrifugal technology to separate the aqueous solution containing medicinal ingredients from the Ganoderma lucidum residue; obtaining the Ganoderma lucidum extract and the Ganoderma lucidum residue, and the Ganoderma lucidum extract is used for other medicinal efficacy research.

[0089] Step S4, soaking and extracting the Ganoderma lucidum residue obtained after centrifugal separation with NaOH solution at a material-liquid ratio of 1:10-1:40, the extraction temperature is 40-100°C, the extraction time is 1-5 hours, wherein the concentration of sodium hydroxide is 0.05-0.5 mol / L, after the extraction is completed, hydrochloric acid is added for neutralization and centrifugation is performed to remove the residue to obtain a mixed solution.

[0090] In step S5, the mixed solution is concentrated and desalted by membrane concentration technology to remove the NaCl component in the mixed solution to obtain a concentrated solution containing the medicinal components.

[0091] Step S6, adding purified water to the freeze-dried powder containing the concentrated active ingredient to prepare the powder to 40-80 mg / ml, and concentrating and freeze-drying the powder after multiple column chromatography separation to obtain Ganoderma lucidum polysaccharide GLP-4.

[0092] The method has simple extraction process, high polysaccharide extraction rate, reuse of Ganoderma lucidum medicinal residue, and low production cost.

[0093] The purpose of the present invention is to provide a Ganoderma lucidum polysaccharide GLP-4, the structural formula of the Ganoderma lucidum polysaccharide GLP-4 is Molecular formula (C 162 H 270 O 135 ) n , where n=10-15.

[0094] n is 10, 11, 12, 13, 14 or 15.

[0095] After obtaining the Ganoderma lucidum polysaccharide GLP-4 with the above structure, we conducted a determination experiment and obtained the following reported results.

[0096] 2. Molecular weight determination

[0097] 1. Purpose of the experiment

[0098] The molecular weight and purity of polysaccharide GLP-4 were determined by HPGPC.

[0099] 2. Experimental Materials

[0100] 2.1 Instruments

[0101] 2.2 Materials

[0102] 2.3 Standards

[0103] 3. Experimental Procedure

[0104] 3.1 Reagent preparation

[0105] 3.2 Preparation of sample and standard solutions

[0106] Accurately weigh the sample and standard, prepare the sample into a 5 mg / ml solution, centrifuge at 12000 rpm for 10 min, filter the supernatant with a 0.22 μm microporous filter membrane, and then transfer the sample to a 1.8 ml injection vial.

[0107] 3.3 Chromatographic method

[0108] Chromatographic column: BRT105-104-102 gel column in series (8×300 mm); mobile phase: 0.05 M NaCl solution; flow rate: 0.6 ml / min, column temperature: 40°C; injection volume: 20 μl; detector: differential detector RI-10A.

[0109] 4. Experimental Results

[0110] As shown in Figure 2-4, the calibration curves of 1gMp-RT (peak molecular weight), 1gMw-RT (weight average molecular weight), and 1gMn-RT (number average molecular weight) were obtained.

[0111] The equation of the 1gMp-RT calibration curve is: y = -0.1796x + 11.518R 2 =0.9957;

[0112] The equation of the lgMw-RT calibration curve is: y = -0.1914x + 12.069R 2 =0.9944;

[0113] The equation of the lgMn-RT calibration curve is: y = -0.1774x + 11.357R 2 =0.9921;

[0114] Based on the standard curve, a calculation formula was derived to calculate the molecular weight of each sample. The molecular weight spectrum of the samples is shown in Figure 5, and the calculation results are shown in the table below.

[0115] Among them, 46.1min is the peak of the mobile phase.

[0116] 2. Monosaccharide composition determination experiment

[0117] 1. Purpose of the experiment

[0118] The monosaccharide composition was determined using ion chromatography.

[0119] 2. Experimental Principle

[0120] Based on the electrochemical activity of carbohydrate molecules and their ionization in strong alkaline solutions, carbohydrate compounds are weak acids with a pKa greater than 11. In high-pH eluents, they exist partially or completely as anions. By taking advantage of the differences in ion exchange caused by the pKa of different carbohydrate compounds and the different hydrophobic interactions between certain carbohydrates and anion exchange resins, efficient anion exchange separation of carbohydrate compounds is achieved. The current generated by the oxidation reaction of hydroxyl groups in the carbohydrate molecular structure on the gold electrode surface is then detected.

[0121] 3. Experimental Materials

[0122] 3.1 Instruments

[0123] 3.2 Reagents

[0124] 3.3 Standards

[0125] 4. Experimental Methods

[0126] 4.1 Reagent preparation

[0127] 4.2 Preparation and calculation method of standard solution

[0128] Sixteen monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid, mannuronic acid) were prepared into standard stock solutions.

[0129] Prepare a standard concentration of each monosaccharide standard solution as a mixed standard. Determine the mass of each monosaccharide using the absolute quantification method, and calculate the molar ratio based on the molar mass of the monosaccharides.

[0130] 4.3 Sample preparation

[0131] Accurately weigh 5 mg of GLP-4 sample into an ampoule, add 2 ml of 3M TFA, and hydrolyze at 120°C for 3 h. Accurately transfer the acid hydrolysis solution to a tube and blow dry with nitrogen. Add 5 ml of water and vortex to mix thoroughly. Aspirate 50 μL and add 950 μL of deionized water. Centrifuge at 12,000 rpm for 5 min. Collect the supernatant for IC analysis.

[0132] 4.4 Chromatographic methods

[0133] Chromatographic column: Dionex Carbopac TM PA20 (3*150mm); mobile phase: A: H2O; B: 15mMNaOHC: 15mMNaOH & 100mM NaOAC; flow rate: 0.3ml / min; injection volume: 5μL; column temperature: 30℃; detector: electrochemical detector.

[0134] 4.5 Standard Sequence

[0135] C(standard) / A(standard)=C(sample) / A(sample)

[0136] 5. Experimental Results

[0137] Mixed standard: Solvent peak: 2.0 min is the peak of sodium hydroxide, 41 min is the peak of sodium acetate, as shown in Figures 6 and 7.

[0138] 3. Ganoderma lucidum polysaccharide GLP-4 linking mode determination experiment

[0139] 1. Purpose of the experiment

[0140] The Ganoderma lucidum polysaccharide GLP-4 sample was derivatized by methylation and then its connection mode was determined by GC-MS.

[0141] 2. Experimental Materials

[0142] 2.1 Instruments

[0143] 2.2 Reagents

[0144] 3. Experimental Methods

[0145] 3.1 Reagent preparation

[0146] 3.2 Sample methylation

[0147] After methylation, hydrolysis and acetylation, the samples were determined by GC-MS and compared with the standard mass spectrum library.

[0148] Weigh 2-3 mg of GLP-4 polysaccharide from Ganoderma lucidum into a glass reaction vial. Add 1 mL of anhydrous DMSO and quickly add methylation reagent solution A. Seal the vial, dissolve under ultrasound, and then add methylation reagent solution B. React in a magnetically stirred water bath at 30°C for 60 minutes. Finally, add 2 mL of ultrapure water to terminate the methylation reaction.

[0149] Take the methylated polysaccharide and add 1 ml of 2 M trifluoroacetic acid (TFA) for hydrolysis for 90 minutes, then evaporate to dryness on a rotary evaporator. Add 2 ml of double-distilled water and 60 mg of sodium borohydride to the residue for reduction for 8 hours, neutralize with glacial acetic acid, rotary evaporate, and oven dry at 101°C. Then, add 1 ml of acetic anhydride for acetylation at 100°C for 1 hour, then cool. Then, add 3 ml of toluene, concentrate under reduced pressure, and evaporate to dryness. Repeat this process 4-5 times to remove excess acetic anhydride.

[0150] The acetylated product was dissolved in 3 mL of CH₂Cl₂ and transferred to a separatory funnel. A small amount of distilled water was added and shaken thoroughly, followed by removal of the upper aqueous layer. This process was repeated four times. The CH₂Cl₂ layer was dried over an appropriate amount of anhydrous sodium sulfate, brought to a volume of 10 mL, and placed in a liquid phase vial. The acetylated product samples were analyzed using a Shimadzu GCMS-QP 2010 gas chromatograph-mass spectrometer.

[0151] GC-MS conditions: RXI-5SIL MS column 30m*0.25mm*0.25um; temperature program conditions: starting temperature 120℃, heating to 250℃ / min at 3℃ / min; hold for 5min; injection port temperature 250℃, detector temperature 250℃, carrier gas helium, flow rate 1mL / min.

[0152] 4. Experimental Results

[0153] The GCMS chromatogram of the sample (PMAA) is shown in Figure 8.

[0154] The results of the analysis of Ganoderma lucidum polysaccharide GLP-4 methylated alditol acetyl ester (PMAA) are shown in the table below and in Figures 9-14.

[0155] 4. NMR analysis spectrum and analysis

[0156] 1. Experimental Materials and Instruments

[0157] Heavy water (D2O, 99.9%) and deuterated acetone were used as internal references; freeze dryer and nuclear magnetic resonance (NMR) instrument were Bruker 600M.

[0158] 2. Experimental Procedure

[0159] Weigh 50 mg of polysaccharide GLP-4 sample, dissolve it in 0.5 ml of heavy water and freeze-dry it. Then, the freeze-dried powder is dissolved in 0.5 ml of heavy water and freeze-dried again. Repeat the above process to fully exchange the active hydrogen. Then, the sample is dissolved in 0.5 ml of heavy water and placed in a 600 MHz nuclear magnetic resonance instrument at room temperature (25°C). 1 H NMR spectrum, 13 C NMR spectrum, DEPT135 one-dimensional spectrum and two-dimensional spectrum.

[0160] 3. Experimental Results

[0161] The hydrogen spectrum signals are mainly concentrated between 3.0 and 5.5 ppm. δ3.2-4.0 ppm is the sugar ring proton signal, and the main terminal proton peaks are δ4.68, 4.67, 4.46, 4.45, 4.44, and 4.43, with the signal peaks concentrated in the 4.3-5.5 ppm region, as shown in Figure 15.

[0162] Carbon spectrum analysis 13C NMR (201 MHz, D2O): The C NMR spectrum signals are mainly concentrated between 60-120 ppm. By observing the C NMR spectrum, we can see that the main anomeric carbon signal peaks are δ104.02, 103.92, 103.91, 103.83, and 103.66. The anomeric carbon region is mainly between δ93 and 105. The δ values ​​are 71.47, 74.99, 69.23, 76.08, 62.09, 76.69, 74.09, 82.09, 76.43, 70.09, 74.61, 76.65, 76.01, 70.89, 70.13, 74.42, 85.43, 69.96, 77.31, 70.13, 74.16, 71.57, 82.28, 76.68, 61.66, 74.76, 85.64, 69.36, 77.11, and 62.03 ppm. Based on the monosaccharide composition, the polysaccharide is composed of glucose. This indicates that the polysaccharide GLP-4 is primarily glucan, as shown in Figure 16.

[0163] Dept 135 spectrum analysis showed that the peaks at 60.70, 68.82, 61.66, 62.04, 70.28, and 68.79 ppm were inverted peaks, indicating chemical shifts at C6, as shown in Figure 17 .

[0164] As shown in Figures 18-21, the anomeric carbon signal is observed to be δ103.94 through HSQC spectra, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ4.68. Through HH-COSY, the signals of H1-2 are 4.68 / 3.44; the signals of H2-3 are 3.44 / 3.65; and the signals of H3-4 are 3.65 / 3.41. We can infer that H1, H2, H3, and H4 are δ4.68, 3.44, 3.65, and 3.41, respectively. The corresponding δ103.66, 74.76, 85.64, and 69.36. The corresponding C5 is 77.11; the chemical shift of C6 is δ62.03. Therefore, this signal should be attributed to the glycosidic bond →3)-β-Glcp-(1→

[0165] Through the HSQC spectrum, it can be observed that the anomeric carbon signal is δ103.91, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ4.44. Through HH-COSY, the signals of H1-2 are 4.44 / 3.23; the signals of H2-3 are 3.23 / 3.41; the signals of H3-4 are 3.41 / 3.55; we can infer that H1, H2, H3, and H4 are δ4.44, 3.23, 3.41, and 3.55, respectively, and their corresponding C1-4 are δ103.92, 74.61, 76.65, and 76.01; and the NOESY spectrum observes that δ4.44 has correlated peaks with 3.23, 3.41, 3.55, and 4.12. Dept135 combined with HSQC can determine that δ3.77 and 4.12 are attributed to the peaks of H6a, b, and H5 is 3.36ppm. The corresponding C5 is 70.89; the chemical shift of C6 is δ70.13, and the corresponding H6a is δ3.77, 4.12. Therefore, this signal should be attributed to the glycosidic bond →6)-β-Glcp-(1→

[0166] Based on similar rules and combined with HMBC and NOESY, all glycosidic bond signals were assigned as shown in the following table:

[0167] Hydrogen and carbon signal assignment

[0168] Main chain analysis:

[0169] In the HMBC spectrum, we assigned the glycosidic bond signals of the polysaccharide GLP-4 based on the one- and two-dimensional NMR spectra. Analysis of the H-spectrum revealed a chemical shift less than 5 ppm, indicating that the monosaccharide is glucose, thus confirming it to be β-glucan. The anomeric hydrogen of the glycosidic bond (→6)-β-D-Glcp-(1→) has a correlated signal peak with its own C6, indicating the presence of a →6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→ linkage.

[0170] The anomeric hydrogen of the glycosidic bond →6)-β-D-Glcp-(1→ and C6 of its →3,6)-β-D-Glcp-(1→) have related signal peaks, indicating the existence of a linking mode of →6)-β-D-Glcp-(1→3,6)-β-D-Glcp-(1→.

[0171] The anomeric hydrogen of the glycosidic bond →3,6)-β-D-Glcp-(1→) has a related signal peak with its own C6, indicating the existence of a linking mode of →3,6)-β-D-Glcp-(1→4,6)-β-D-Glcp-(1→.

[0172] The anomeric hydrogen of the glycosidic bond →3,6)-β-D-Glcp-(1→ and C6 of its →4,6)-β-D-Glcp-(1→) have related signal peaks, indicating the existence of the →3,6)-β-D-Glcp-(1→4,6)-β-D-Glcp-(1→ linking mode.

[0173] Branch chain analysis:

[0174] In the NOESY spectrum, the anomeric hydrogen of the glycosidic bond β-D-Glcp-(1→) and H3 of its →3)-β-D-Glcp-(1→) have related signal peaks, indicating the existence of a linkage mode of β-D-Glcp-(1→3)-β-D-Glcp-(1→.

[0175] The anomeric hydrogen of the glycosidic bond →3)-β-D-Glcp-(1→ and its H3 of →3)-β-D-Glcp-(1→ have related signal peaks, indicating the existence of the linking mode of β-D-Glcp-(1→3)-β-D-Glcp-(1→.

[0176] The anomeric hydrogen of the glycosidic bond →3)-β-D-Glcp-(1→ and H4 of its →3,6)-β-D-Glcp-(1→ have related signal peaks, indicating the existence of the →3)-β-D-Glcp-(1→3,6)-β-D-Glcp-(1→ linking mode.

[0177] The anomeric hydrogen of the glycosidic bond →4)-β-D-Glcp-(1→ and its H4 of →4,6)-β-D-Glcp-(1→ have related signal peaks, indicating the existence of the linking mode of →4)-β-D-Glcp-(1→4,6)-β-D-Glcp-(1→

[0178] In summary, we can infer that the main chain of the polysaccharide GLP-4 is β-1,6 glucan, and β-D-Glcp-(1→3)-β-D-Glcp-(1→) and β-D-Glcp-(1→4)-β-D-Glcp-(1→) are connected to the main chain through the O-3 of →3,6)-β-D-Glcp-(1→) and the O-4 of →4,6)-β-D-Glcp-(1→), respectively. The simplified molecular formula is shown below.

[0179] Another object of the present invention is to provide an application of GLP-4, a polysaccharide of Ganoderma lucidum. The polysaccharide GLP-4 of Ganoderma lucidum has good water solubility, is easily absorbed by the human body, and has therapeutic effects on emphysema, pulmonary fibrosis and tumors. In particular, the therapeutic effect on emphysema and pulmonary fibrosis is extremely excellent, which is significantly better than pirfenidone. When used in combination with chemotherapy drugs, it can eliminate the toxic side effects of chemotherapy drugs on the human body and has a certain effect on controlling and reducing tumor masses and reducing and eliminating tumor cells.

[0180] The anti-tumor effect of GLP-4 is shown in the following examples:

[0181] Experimental study and data on the anti-tumor effect of Ganoderma lucidum polysaccharide GIP-4 combined with chemotherapy on LLC lung cancer-bearing mice

[0182] Purpose of the experiment

[0183] Lewis lung cancer cells were inoculated in the right axilla of C57 mice to prepare tumor-bearing mice. The anti-tumor effect of Ganoderma lucidum polysaccharide GLP-4 on lung cancer-bearing mice was studied to provide experimental basis for its clinical research.

[0184] Experimental Materials

[0185] Test sample

[0186] Ganoderma lucidum polysaccharide GLP-4, batch number: OLMJT202108(2-4), was provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0187] Positive control

[0188] Kanglaite soft capsules, batch number: 20200802, product of Zhejiang Kanglaite Pharmaceutical Co., Ltd.; cisplatin injection, batch number: 601210104, product of Jiangsu Hausen Pharmaceutical Group Co., Ltd.

[0189] experimental animals

[0190] 80 SPF male C57 mice, weighing 12-15 g, were provided by Hunan Slake Jingda Experimental Animal Co., Ltd., with experimental animal production license number: SCXK (Xiang) 2019-0004 and experimental animal quality certificate number: 430727211103153225. They were raised in Area D of the barrier environment animal laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd., with experimental animal use license number: SYXK (Xiang) 2020-0015.

[0191] Main reagents

[0192] 0.9% sodium chloride injection, batch number: 21071401C, product of Hunan Kangyuan Pharmaceutical Co., Ltd.; ALP assay kit, batch number: 201223, purchased from Mike Biotech Co., Ltd.; TP assay kit, batch number: 105467, ALB assay kit, batch number: 105468, ALT assay kit, batch number: 105475, AST assay kit, batch number: 104446, TBIL assay kit, batch number: 105465, GGT assay kit, batch number: 104447, all are products of Wako Pure Chemical Industries, Ltd.

[0193] Main instruments

[0194] S10 portable high-speed disperser, Shanghai Xinzhi; AR223CN electronic balance, Ohaus Instruments (Changzhou) Co., Ltd.; LABOSPECT003 automatic biochemical analyzer, Hitachi, Japan; TDZ5-WS desktop multi-tube automatic balancing centrifuge, Hunan Kaida Industrial Development Co., Ltd.; ME2002E electronic balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.; digital display vernier caliper, Yongkang Zhengfeng Hardware Co., Ltd.; flow cytometer, BD; ASP200S fully automatic tissue dehydrator, ASP300S fully automatic tissue dehydrator, TP1020 fully automatic dehydrator, HI1210 slide spreader, HI1220 slide baking machine, RM2235 paraffin slicer, EG1150H+C tissue embedding machine, AutoStainer XL automatic slide stainer + CV5030 automatic coverslip machine, Leica, Germany; BX43 biological microscope + MD50 digital imaging system, CX31 biological microscope, Olympus, Japan.

[0195] Experimental methods

[0196] 1×10 Lewis tumor cells in logarithmic growth phase were taken 7 0.2 mL of the solution was inoculated into the right axilla of 10 pre-purchased normal male C57 mice. The tumor growth was observed and the mice were prepared to bear tumors. When the tumor volume of the mice reached 200 mm 3 At the above time, the tumor tissue of the tumor-bearing mice was aseptically removed to prepare a tissue cell suspension, and the tumor tissue homogenate was made with cell culture medium at a ratio of 1:1 (V:V). The suspension was inoculated into the right axilla of 60 male C57 mice with an inoculation volume of 0.2 mL per mouse. The suspension was kept for 24 h until the tumor volume of the tumor-bearing mice reached 100 mm. 3 At the above time, tumor-bearing mice with no ulceration and good growth were selected and randomly divided into groups according to the tumor volume, namely: model control group, cisplatin group (4 mg / kg), Kanglaite soft capsule group (1404 mg / kg), cisplatin + Kanglaite soft capsule group (4+1404 mg / kg), cisplatin + GLP-4 low-dose group (4+130 mg / kg), cisplatin + GLP-4 high-dose group (4+1170 mg / kg), 8 mice in each group, and another 8 mice were taken as normal control group. The mice in the cisplatin group were intraperitoneally injected with cisplatin solution, and the mice in the other groups were given the corresponding solution, with the dosage volume of 20 mL / kg (oral gavage) and 10 mL / kg (intraperitoneal injection), respectively, once a day, for 27 consecutive days. After the last dose, orbital blood was collected to detect blood WBC, RBC, liver and kidney function (ALT, AST, BUN, CRE) and CD3 + 、CD4 + 、CD8 +The content was determined, bone marrow was taken for smear examination, spleen, thymus and tumor were taken for weighing and tumor, spleen, stomach and kidney were subjected to histopathological examination.

[0197] Dosage design

[0198] Ganoderma lucidum polysaccharide GLP-4 was designed based on the results of previous studies. The low dose was 130 mg / kg and the high dose was 1170 mg / kg. The corresponding drugs were administered as shown in Table 1. This experiment was conducted at the same multiple of the intended clinical dose.

[0199] The proposed clinical dosage of Kanglaite Soft Capsules is 0.45g / capsule, 6 capsules / time, 4 times / day, which is 10.8g / day. The equivalent dose in mice based on body surface area is 10.8g / day*0.0026 / 0.02kg=1404mg / kg. This trial was conducted at the same multiple of the proposed clinical dosage.

[0200] Cisplatin dosage design basis: According to the clinical dosage of cisplatin, each person should not exceed 100 mg / m2 per day. 2 The tolerance of mice to cisplatin was evaluated, and a dose of 4 mg / kg was selected as the administration dose.

[0201] Table 1 Trial grouping and dosage design

[0202] Detection indicators

[0203] Efficacy indicators

[0204] Evaluation of animal quality of life and survival time: During the experiment, the weight of each group of animals, animal mortality, mental state, coat color, coat smoothness, stool form, and the presence of diarrhea were recorded in detail.

[0205] Tumor volume detection: After administration, the length and width of the mouse tumor were measured with a vernier caliper every 3 days to calculate the tumor volume. Tumor volume (TV) = 1 / 2 × a (length) × b 2 (width). Relative tumor volume (RTV) = TV t / TV0, TV0 is the tumor volume at the time of drug administration, TV t is the tumor volume measured each time. Relative tumor proliferation rate T / C (%) = T RTV / C RTV ×100, T RTV :RTV in treatment group; C RTV : RTV of model control group. Tumor growth inhibition rate (%) = (tumor weight of model control group - tumor weight of drug-treated group) / tumor weight of model control group × 100%. A tumor growth inhibition rate (%) < 40% was considered ineffective; a tumor growth inhibition rate (%) ≥ 40% and P ≤ 0.05 was considered effective.

[0206] Spleen and thymus organ coefficients: After the last administration, the spleen, thymus, and tumor were weighed and the organ coefficients were calculated. Organ coefficient (%) = organ weight / fasting body weight × 100%.

[0207] Reduce toxicity and increase efficacy

[0208] Hematological tests: After the last dose, blood was collected from the orbital cavity for routine blood tests (WBC, RBC) and liver and kidney function indicators (BUN, CRE).

[0209] Bone marrow examination: After the last administration, bone marrow was taken for smear and the changes of bone marrow cells in each group of mice were observed.

[0210] Histopathological examination: After the last administration, tumor tissues, spleen, stomach, and kidney were collected for HE staining to observe the histopathological changes.

[0211] Blood CD4 + 、CD8 + Content detection: After the last administration, blood lymphocyte typing CD4 was detected by flow cytometry + , CD8 + content.

[0212] Data processing and statistical analysis

[0213] The significant figures of the experimental data were rounded off and statistical analysis was performed in accordance with the SOP of this center. The statistical software used was SPSS. The measurement data were expressed as mean ± standard deviation. Normality and homogeneity of variance were tested using the Leven's test. If there was no statistical significance (P>0.05), one-way analysis of variance (ANOVA) was used for statistical analysis. If the ANOVA was statistically significant (P≤0.05), the LSD test (parametric method) was used for comparative analysis. If the variances were unequal (P≤0.05), the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P≤0.05), the Dunnett's test (nonparametric method) was used for comparative analysis. Statistical results were tested with an α=0.05 threshold, where P≤0.05 indicated statistical significance and P≤0.01 indicated that the difference being tested was highly significant.

[0214] Experimental results

[0215] General clinical observations and animal deaths

[0216] Before administration, mice were active, with normal movements and gait. Model control group mice 2M06 and 2M03 died on January 20, 2022, and January 21, 2022, respectively; Kanglaite soft capsule group mice 4M01 died on January 19, 2022; cisplatin + Kanglaite soft capsule group mice 5M01 and 5M04 died on January 14, 2022, and January 22, 2022, respectively; cisplatin + GLP-4 low-dose group mice 8M06 / 08 and 8M05 / 07 died on January 12, 2022, and January 15, 2022, respectively; and cisplatin + GLP-4 high-dose group mice 9M05 / 06 and 9M08 died on January 8, 2022, and January 23, 2022, respectively.

[0217] As shown in Table 2 , the mortality rates of each group were 0%, 25.0%, 0%, 12.5%, 25.0%, 50.0%, and 37.5%, respectively.

[0218] Table 2 Statistics of the number of surviving animals and mortality rates in each group

[0219] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on body weight in Lewis lung cancer-bearing mice

[0220] As shown in Table 3, compared with the normal control group, the body weight of mice in the model control group was significantly reduced on Day 4 after administration (P ≤ 0.05). Compared with the model control group, the body weight of mice in the cisplatin group and the cisplatin combined with Kanglaite soft capsule group was significantly reduced on Day 7 to Day 25 after administration (P ≤ 0.01); the body weight of mice in the cisplatin combined with GLP-4 low- and high-dose groups was significantly reduced on Day 4 to Day 25 after administration (P ≤ 0.05 or P ≤ 0.01); and the body weight of mice in the Kanglaite soft capsule group was significantly reduced on Day 10 to Day 19 after administration. Compared with the cisplatin group, the body weight of mice in the Kanglaite soft capsule group was significantly increased on Day 7 to Day 25 after administration (P ≤ 0.05 or P ≤ 0.01), the body weight of mice in the cisplatin combined with GLP-4 high-dose group was significantly reduced on Day 7 after administration (P ≤ 0.05), and the body weight of mice in the cisplatin combined with GLP-4 low-dose group was significantly reduced on Day 7 to Day 13 after administration (P ≤ 0.05 or P ≤ 0.01). Compared with the Kanglaite soft capsule group, the body weight of mice in the cisplatin combined with Kanglaite soft capsule group and the low- and high-dose GLP-4 groups decreased significantly from Day 7 to Day 25 (P ≤ 0.05 or P ≤ 0.01). Compared with the cisplatin + Kanglaite soft capsule group, the body weight of mice in the cisplatin combined with low-dose GLP-4 group decreased significantly from Day 7 to Day 13 (P ≤ 0.05 or P ≤ 0.01), while no statistically significant differences were found in the other groups.

[0221] Table 3 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the body weight of Lewis cell tumor-bearing mice

[0222] Note: Compared with the normal control group, + P≤0.05; compared with the model control group, *P≤0.05, **P≤0.01; compared with the cisplatin group, # P≤0.05, ## P≤0.01; compared with Kanglaite soft capsule group, & P≤0.05, && P≤0.01, compared with cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0223] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on tumor volume and relative tumor volume in Lewis lung cancer-bearing mice

[0224] As shown in Table 4, compared with the model control group, the tumor volume of mice in the high-dose cisplatin combined with GLP-4 group was significantly reduced from D10 to D25 (P≤0.05 or P≤0.01), the tumor volume of mice in the low-dose cisplatin combined with GLP-4 group was significantly reduced from D4 and D10 to D25 (P≤0.05 or P≤0.01), the tumor volume of mice in the cisplatin group was significantly reduced from D10 and D16 to D25 (P≤0.05 or P≤0.01), and the tumor volume of mice in the cisplatin combined with Kanglaite soft capsule group was significantly reduced from D13 to D25 (P≤0.05 or P≤0.01). Compared with the cisplatin group, the tumor volume of mice in the low-dose cisplatin combined with GLP-4 group was significantly reduced from D4 to D16 (P≤0.05), and the tumor volume of mice in the Kanglaite soft capsule group was significantly increased from D7 to D25 (P≤0.05 or P≤0.01). Compared with the Kanglaite soft capsule group, the tumor volume of mice in the high-dose cisplatin combined with GLP-4 group and the Kanglaite soft capsule group was significantly reduced on D13 to D25 (P≤0.05 or P≤0.01), and the tumor volume of mice in the low-dose cisplatin combined with GLP-4 group was significantly reduced on D4 to D7 and D13 to D25 (P≤0.05 or P≤0.01).

[0225] As shown in Table 5, compared with the model control group, the relative tumor volumes of mice in the cisplatin group, the cisplatin combined with Kanglaite soft capsule group, and the cisplatin combined with GLP-4 low- and high-dose groups were significantly reduced from D13 to D25 (P ≤ 0.05 or P ≤ 0.01). Compared with the cisplatin + Kanglaite soft capsule group, the tumor volumes of mice in the cisplatin combined with GLP-4 low-dose group were significantly reduced from D4 to D13 (P ≤ 0.05 or P ≤ 0.01), while no statistical significance was observed in the other groups.

[0226] Table 4 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on tumor volume in Lewis lung cancer-bearing mice

[0227] Note: Compared with the model control group, *P≤0.05, **P≤0.01; compared with the cisplatin group, # P≤0.05, ## P≤0.01; compared with Kanglaite soft capsule group, & P≤0.05, && P≤0.01, compared with cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0228] Table 5 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the relative tumor volume in Lewis cell tumor-bearing mice

[0229] Note: Compared with the model control group, *P≤0.05, **P≤0.01.

[0230] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the relative tumor proliferation rate (T / C) in Lewis lung cancer-bearing mice

[0231] As shown in Table 6, the T / C administration rates of mice in the low-dose cisplatin + GLP-4 group from D4 to D25 were 42.0%, 27.3%, 3.0%, 0.0%, 0.0%, 0.8%, 1.8%, and 2.1%, respectively, and all values ​​were ≤ 60.0%; the T / C administration rates of mice in the high-dose cisplatin + GLP-4 group from D4 to D25 were 57.1%, 27.3%, 3.0%, 0.0%, 0.0%, 2.7%, 4.0%, and 2.1%, respectively. The T / C rates of the mice in the cisplatin group from D7 to D10 and D16 to D25 were 45.5%, 27.3%, 28.2%, 36.9%, 30.9% and 23.6%, respectively, and the values ​​were all ≤60.0%; the T / C rates of the mice in the cisplatin + Kanglaite soft capsule group from D13 to D25 were 7.3%, 3.3%, 15.6%, 7.3% and 15.6%, respectively, and the values ​​were all ≤60.0%.

[0232] Table 6 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the relative tumor growth rate in Lewis lung cancer-bearing mice

[0233] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on organ coefficients and tumor growth inhibition rate in Lewis lung cancer-bearing mice

[0234] As shown in Table 7, compared with the normal control group, the spleen and tumor coefficients in the model control group were significantly increased (P ≤ 0.01), and the thymus coefficient was significantly decreased (P ≤ 0.01). Compared with the model control group, the spleen, thymus, and tumor coefficients of mice in the cisplatin group, the low-dose cisplatin combined with GLP-4 group, and the Kanglaite soft capsule group were significantly decreased (P ≤ 0.05 or P ≤ 0.01), and the spleen and tumor coefficients of mice in the high-dose cisplatin combined with GLP-4 group were significantly decreased (P ≤ 0.01). Compared with the cisplatin group, the spleen and tumor coefficients of mice in the Kanglaite soft capsule group were significantly increased (P ≤ 0.01). Compared with the Kanglaite soft capsule group, the spleen and tumor coefficients of mice in the high-dose cisplatin combined with GLP-4 group were significantly decreased (P ≤ 0.01), and the spleen, thymus, and tumor coefficients of mice in the low-dose cisplatin combined with GLP-4 group were significantly decreased (P ≤ 0.05 or P ≤ 0.01). Compared with the cisplatin + Kanglaite soft capsule group, the organ-to-body ratios of each group had no statistical significance.

[0235] The tumor growth inhibition rate of the Kanglaite soft capsule group was -6.8%, and the values ​​were all <40.0%; the tumor growth inhibition rates of mice in the cisplatin group, cisplatin combined with Kanglaite soft capsule group, and cisplatin combined with GLP-4 low- and high-dose groups were 61.6%, 78.3%, 88.6%, and 94.2%, respectively, and the values ​​were all ≥40.0%.

[0236] Table 7 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on organ coefficients and tumor growth inhibition rate in Lewis lung cancer-bearing mice ( n=4~8)

[0237] Note: Compared with the normal control group, ++ P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01; compared with the cisplatin group, # P≤0.05, ## P≤0.01; compared with Kanglaite soft capsule group, & P≤0.05, && P≤0.01; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0238] In Figure 22, the control groups are marked as follows: 1: model control group, 2: cisplatin group, 3: Kanglaite soft capsule group, 4: cisplatin + Kanglaite soft capsule group, 7: cisplatin + GLP-4 low-dose group, 8: cisplatin + GLP-4 high-dose group.

[0239] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on blood biochemical parameters in Lewis lung cancer-bearing mice

[0240] As shown in Table 8, compared with the normal control group, the blood WBC, ALT, AST, and BUN levels of mice in the model control group were significantly increased (P ≤ 0.05 or P ≤ 0.01), while the RBC levels were significantly decreased (P ≤ 0.05). Compared with the model control group, the blood WBC and AST levels of mice in the low- and high-dose cisplatin combined with GLP-4 groups were significantly decreased (P ≤ 0.05 or P ≤ 0.01). The WBC levels in both the cisplatin group and the cisplatin combined with Kanglaite soft capsule group were significantly decreased (P ≤ 0.05). Compared with the cisplatin group, the blood BUN levels of mice in the Kanglaite soft capsule group were significantly increased (P ≤ 0.05), while the blood RBC levels of mice in the cisplatin combined with Kanglaite soft capsule group were significantly decreased (P ≤ 0.05). Compared with the Kanglaite soft capsule group, the blood WBC levels of mice in the cisplatin combined with Kanglaite soft capsule group were significantly decreased (P ≤ 0.05). Compared with the cisplatin + Kanglaite soft capsule group, there was no statistically significant difference between the groups.

[0241] Table 8 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on blood biochemical indicators in Lewis lung cancer-bearing mice

[0242] Note: Compared with the normal control group, + P≤0.05, ++ P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01; compared with the cisplatin group, # P≤0.05, ## P≤0.01; compared with Kanglaite soft capsule group, & P≤0.05; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05.

[0243] Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on CD3 in Lewis lung cancer-bearing mice + / CD4 + 、CD3 + / CD8 + Impact

[0244] As shown in Table 9, compared with the normal control group, the blood CD3 + / CD8 + Compared with the model control group, the CD3 + / CD4 + Compared with the cisplatin group, the CD3 + / CD4 + 、CD3 + / CD8 +The CD3 + / CD8 + Compared with the Kanglaite soft capsule group, the CD3 + / CD4 + The CD3 + / CD8 + Compared with the cisplatin + Kanglaite soft capsule group, the cisplatin combined with GLP-4 low-dose group had a significant increase in blood CD3 + / CD4 + 、CD3 + / CD8 + The results of the other drug groups were not statistically significant.

[0245] Table 9 Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on CD3 + / CD4 + 、CD3 + / CD8 + Effect of content

[0246] Note: Compared with the normal control group, + P≤0.05; compared with the model control group, *P≤0.05; compared with the cisplatin group, # P≤0.05; compared with Kanglaite soft capsule group, & P≤0.05, && P≤0.01; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0247] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on bone marrow cells in Lewis lung cancer-bearing mice

[0248] As shown in Table 10, compared with the normal control group, the model control group had a significant increase in the monocyte system and other cells in the bone marrow (P ≤ 0.05), an increasing trend in the granulocyte-erythrocyte system and the granulocyte-erythrocyte ratio, and a decreasing trend in the erythrocyte system and lymphocyte system, but these trends were not statistically significant. Compared with the model control group, the bone marrow erythrocyte system of mice in the cisplatin combined with Kanglaite soft capsule group was significantly increased (P ≤ 0.05), while the granulocyte-erythrocyte ratio and the monocyte system were significantly decreased (P ≤ 0.05). The bone marrow monocyte system of mice in the Kanglaite soft capsule group and the cisplatin combined with GLP-4 low-dose group was significantly decreased (P ≤ 0.01). The bone marrow monocyte system and other cells of mice in the cisplatin combined with GLP-4 high-dose group were significantly decreased (P ≤ 0.05 or P ≤ 0.01). Compared with the cisplatin group, the bone marrow monocyte system of mice in the cisplatin combined with GLP-4 low- and high-dose groups was significantly decreased (P ≤ 0.05). Compared with the Kanglaite soft capsule group, the high-dose cisplatin combined with GLP-4 group had significantly decreased other bone marrow cells (P ≤ 0.05). Compared with the cisplatin + Kanglaite soft capsule group, the low-dose cisplatin combined with GLP-4 group had significantly increased bone marrow granulocyte-erythrocyte system and granulocyte-erythrocyte ratio (P ≤ 0.05), while the other groups showed no statistically significant difference.

[0249] Table 10 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on bone marrow cells in Lewis lung cancer-bearing mice

[0250] Note: Compared with the normal control group, + P≤0.05; compared with the model control group, *P≤0.05, **P≤0.01; compared with the cisplatin group, # P≤0.05, ## P≤0.01; compared with Kanglaite soft capsule group, & P≤0.05, && P≤0.01; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0251] Effects of Ganoderma lucidum polysaccharide GL-4 combined with chemotherapy on tumor, spleen, kidney and stomach tissue pathology in Lewis cell tumor-bearing mice

[0252] As shown in Figure 23, the model control group showed abundant tumor cells surrounding the tumor, with densely arranged cells, large cell size, a high nuclear-cytoplasmic ratio, and frequent mitotic figures. In the cisplatin group, extensive necrosis and hemorrhage were observed in the tumor center, and the number of tumor cells was abundant. Necrosis was observed in the tumor center of the cisplatin + Kanglaite Soft Capsule group and the cisplatin + GLP-4 low-dose group, and the number of surrounding tumor cells decreased. Vacuoles were observed in the tumor tissue of the cisplatin + GLP-4 high-dose group.

[0253] As shown in Figures 24 and 25, no obvious abnormalities were found in the kidney and stomach pathology of each group.

[0254] As shown in Figure 26, the model control group showed obvious red pulp hematopoiesis with diverse cell types. The cisplatin + Kanglaite soft capsule group showed no obvious abnormalities in the spleen, while the spleens of the other groups showed varying degrees of red pulp hematopoiesis with diverse cell types.

[0255] In Figures 23-26, the control groups are marked as follows: A: normal group; B: model control group; C: cisplatin group; D: Kanglaite soft capsule group; E: cisplatin + Kanglaite soft capsule group; H: cisplatin + GLP-4 low-dose group; I: cisplatin + GLP-4 high-dose group

[0256] in conclusion

[0257] Ganoderma lucidum polysaccharide GLP-4 combined with cisplatin can significantly inhibit the growth of tumors in LLC lung cancer-bearing mice and has a significant synergistic effect.

[0258] Experimental study and data on the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 liver cancer-bearing mice

[0259] Purpose of the experiment

[0260] The H22 liver cancer homogenate was inoculated into the right axilla of C57 mice to prepare tumor-bearing mice. The effect of Ganoderma lucidum polysaccharide GLP-4 on liver cancer-bearing mice was studied to provide experimental basis for its clinical research.

[0261] Experimental Materials

[0262] Test sample

[0263] Ganoderma lucidum polysaccharide GLP-4, batch number: OMLJT202108(2-4), was provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0264] Positive control

[0265] Cisplatin, batch number: E21268081, product of Shanghai Aladdin Biochemical Technology Co., Ltd.

[0266] experimental animals

[0267] Seventy SPF male C57 mice, weighing 14–16 g, were provided by the Guangdong Medical Laboratory Animal Center. The laboratory animal production license number is SCXK (Guangdong) 2022-0002, and the laboratory animal quality certificate number is 44007200111236.

[0268] Main reagents

[0269] PBS buffer was prepared by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.; fetal bovine serum was a product of Zhejiang Tianhang Biotechnology Co., Ltd.; 1640 culture medium was a product of Gibco; and 0.25% trypsin was a product of Gibco.

[0270] Main instruments

[0271] Vernier caliper, product of Shanghai Tool Factory Co., Ltd.; I-2000 balance, product of Dongguan Nancheng Changxie Electronic Products Factory; ophthalmic scissors and tweezers, product of Shanghai Jinzhong Medical Instrument Co., Ltd.; CCL-170B-8 carbon dioxide incubator, product of Singapore ESCO; Luna-Ⅱ cell counter, product of Nanjing Hengqiao Instrument Co., Ltd.; JRA-35S handheld homogenizer, product of Wuxi Jerian Instrument Equipment Co., Ltd.

[0272] Experimental methods

[0273] The H22 liver cancer solid tumor homogenate suspension was injected subcutaneously into the axilla of 60 healthy male C57 mice to establish a solid tumor model. 3 At approximately 60 days, mice were randomly divided into groups based on tumor volume and given the corresponding drug or vehicle by gavage or intraperitoneal injection for 27 consecutive days. Tumor length and short diameter were measured every 3 days to calculate tumor volume, and mice were weighed every 3 days. At the end of the experiment, tumors, spleens, and thymuses were removed and weighed, and the tumor index, spleen index, and thymus index were calculated.

[0274] Dosage design

[0275] Ganoderma lucidum polysaccharide GLP-4 was designed based on previous experimental results. The low dose was 50 mg / kg and the high dose was 150 mg / kg. The corresponding drugs were administered as shown in Table 11.

[0276] Cisplatin dosage design basis: According to the clinical dosage of cisplatin, each person should not exceed 100 mg / m2 per day. 2 The tolerance of mice to cisplatin was evaluated and a dose of 3 mg / kg was selected as the administration dose.

[0277] Table 11 Trial grouping and dosage design

[0278] Detection indicators

[0279] Efficacy indicators

[0280] Relative tumor inhibition rate

[0281] Relative tumor inhibition rate (%) = (1-T RTV / C RTV )×100%. Among them, TRTV is the relative tumor volume of the experimental group, C RTV is the relative tumor volume of the model control group. t / V0,V t =V0 is the tumor volume of mice on day t of drug administration, and V0 is the tumor volume of mice at the time of grouping. Evaluation criteria: Relative tumor inhibition rate ≥ 40%, and P < 0.05 after statistical analysis is considered an effective inhibition level.

[0282] Tumor growth inhibition rate

[0283] Tumor growth inhibition rate (%) = (1-T / C) × 100%. Where T represents the average tumor weight of the treatment group and C represents the average tumor weight of the model control group. Evaluation criteria: Tumor growth inhibition rate ≥ 40% and statistical analysis P < 0.05 are considered effective inhibition levels.

[0284] Spleen and thymus organ coefficients: After the last administration, the spleen, thymus, and tumor were weighed and the organ coefficients were calculated.

[0285] Tumor index (%) = tumor weight / body weight × 100%.

[0286] Immune organ index (mg / g) = organ mass / body weight × 1000.

[0287] Data processing and statistical analysis

[0288] SPSS 17.0 was used for statistical analysis, and the statistical significance level was set at P ≤ 0.05. The measurement data were expressed as mean ± standard deviation. Leven's test was used to test normality and homogeneity of variance. If normality and homogeneity of variance were met (P>0.05), one-way analysis of variance (ANOVA) and LSD test were used for statistical analysis. If normality and homogeneity of variance were not met (P<0.05), Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P<0.05), Dunnett's Test (non-parametric method) was used for comparative analysis. Statistical differences and biological significance were considered in the evaluation.

[0289] Experimental results

[0290] Animal deaths

[0291] As shown in Table 12, the mortality rate of mice in each group was 0.

[0292] Table 12 Statistics of the number of surviving animals and mortality rates in each group

[0293] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on body weight in H22 liver cancer-bearing mice

[0294] As shown in Table 13, compared with the normal group, the body weight of mice in the model control group increased significantly from D0 to D27; the body weight of mice in the cisplatin group increased significantly from D0 and D3, and decreased significantly from D15 to D27; the body weight of mice in the cisplatin + GLP-4 low-dose group decreased significantly from D12 to D27; and the body weight of mice in the cisplatin + GLP-4 high-dose group decreased significantly from D9 to D27.

[0295] Compared with the model control group, the body weight of mice in the cisplatin group was significantly reduced from D6 to D27, and the body weight of mice in the cisplatin + GLP-4 low-dose and high-dose groups was significantly reduced from D3 to D27.

[0296] Compared with the cisplatin group, the body weight of mice in the cisplatin + GLP-4 high-dose group was significantly reduced on D15 and D24.

[0297] Table 13 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on body weight of H22 liver cancer-bearing mice

[0298] Note: Compared with the model control group, + P<0.05; compared with the cisplatin group, # P<0.05; compared with the normal group, * P<0.05.

[0299] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on tumor volume in H22 liver cancer-bearing mice

[0300] As shown in Table 14, compared with the model control group, the tumor volume of the cisplatin group was significantly reduced from D9 to D27, the tumor volume of the cisplatin + GLP-4 low-dose group was significantly reduced from D6 to D27, and the tumor volume of the cisplatin + GLP-4 high-dose group was significantly reduced from D9 to D27.

[0301] Compared with the cisplatin group, the tumor volume of the cisplatin + GLP-4 low-dose group was significantly reduced on D12 to D18 and D27.

[0302] Table 14 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on tumor volume in H22 liver cancer-bearing mice

[0303] Note: Compared with the model control group, + P<0.05; compared with the cisplatin group, # P<0.05.

[0304] As shown in Tables 15 and 16, compared with the model control group, the relative tumor inhibition rates of the cisplatin group on D9 to D27 were above 60%, namely 66.5%, 70.6%, 69.1%, 81.3%, 83.9%, 85.3%, and 84.3%, respectively (P < 0.05); the relative tumor inhibition rates of the cisplatin + GLP-4 low-dose group on D6 to D27 were above 55%, respectively 56.1% and 53.9%, respectively. %, 77.1%, 83.6%, 87.9%, 91.7%, 91.2%, 91.1%, 94.2% (P<0.05); the relative tumor inhibition rates of the high-dose cisplatin + GLP-4 group were above 60% on D9 to D27, which were 65.2%, 76.6%, 81.9%, 85.8%, 86.4%, 88.7%, 90.9% (P<0.05).

[0305] Compared with the cisplatin group, the relative tumor inhibition rates of the low-dose cisplatin + GLP-4 group on D12 to D18 and D27 were 44.4%, 60.9%, 55.6%, and 63.3%, respectively (P<0.05); the relative tumor inhibition rates of the high-dose cisplatin + GLP-4 group on D15 and D27 were 41.6% and 42.3%, respectively.

[0306] Table 15 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the relative tumor inhibition rate of H22 liver cancer-bearing mice (compared with the model control group)

[0307] Note: Compared with the model control group, + P<0.05.

[0308] Table 16 Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the relative tumor inhibition rate of H22 liver cancer-bearing mice (compared with the cisplatin group)

[0309] Note: Compared with cisplatin group, # P<0.05.

[0310] As shown in Table 17, compared with the model group, the tumor index, spleen index, and thymus index were significantly decreased in the cisplatin group, the low-dose cisplatin + GLP-4 group, and the high-dose cisplatin + GLP-4 group. Compared with the cisplatin group, the thymus index in the low-dose cisplatin + GLP-4 group was significantly decreased. Compared with the normal group, the spleen index in the model control group was significantly increased, while the thymus index in the cisplatin group, the low-dose cisplatin + GLP-4 group, and the high-dose cisplatin + GLP-4 group was significantly decreased.

[0311] Compared with the model control group, the tumor growth inhibition rates in the cisplatin group were 88.7%, and those in the low-dose cisplatin + GLP-4 group and the high-dose group were 91.5% and 88.2%, respectively. Compared with the cisplatin group, the tumor growth inhibition rates in the low-dose cisplatin + GLP-4 group and the high-dose cisplatin + GLP-4 group were 25.1% and -4.4%, respectively.

[0312] Table 17 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on organ index and tumor growth inhibition rate in H22 liver cancer-bearing mice

[0313] Note: Compared with the model control group, + P<0.05; compared with the cisplatin group, # P<0.05; compared with the normal group, * P<0.05.

[0314] As shown in Figures 27-30, these are pictures of tumor-bearing mice, and the corresponding groups are: Figure 27: Model control group, Figure 28: Cisplatin group, Figure 29: Cisplatin + GLP-4 low-dose group, Figure 30: Cisplatin + GLP-4 high-dose group

[0315] Figures 31-34 show tumor images of tumor-bearing mice, and the corresponding groups are: Figure 31: Model control group, Figure 32: Cisplatin group, Figure 33: Cisplatin + GLP-4 low-dose group, Figure 34: Cisplatin + GLP-4 high-dose group

[0316] in conclusion

[0317] Ganoderma lucidum polysaccharide GLP-4 combined with cisplatin can significantly inhibit the growth of tumors in H22 liver cancer-bearing mice and has a significant synergistic effect

[0318] Experimental study and data on the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 orthotopic liver cancer in mice

[0319] Purpose of the experiment

[0320] In this study, H22 liver cancer cells were inoculated in the right axilla of C57 mice to prepare mice with orthotopic tumors. The anti-tumor effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 orthotopic liver cancer mice was studied to provide experimental basis for its clinical research.

[0321] Experimental Materials

[0322] Test sample

[0323] Ganoderma lucidum polysaccharide GLP-4, batch number: OLMJT202108(2-4), was provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0324] Reference substances

[0325] Kanglaite soft capsules, batch number: 20211006, product of Zhejiang Kanglaite Pharmaceutical Co., Ltd.; cisplatin injection, batch number: 601211204, product of Jiangsu Hausen Pharmaceutical Group Co., Ltd.

[0326] experimental animals

[0327] Sixty-five SPF male C57 mice, weighing 12–15 g, were provided by Hunan Slake Jingda Experimental Animal Co., Ltd. (Laboratory Animal Production License No.: SCXK (Xiang) 2019-0004) and raised in Area D of the Barrier Environment Animal Laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd. (Laboratory Animal Use License No.: SYXK (Xiang) 2020-0015).

[0328] Main reagents

[0329] 0.9% sodium chloride injection, batch number: 21071401C, product of Hunan Kangyuan Pharmaceutical Co., Ltd.; ALT assay kit, batch number: 201751, AST assay kit, batch number: 110620, CRE assay kit, batch number: 111644, BUN assay kit, batch number: 201749, all are products of Wako Pure Chemical Industries, Ltd. of Japan.

[0330] Main instruments

[0331] AR223CN electronic balance, Ohaus Instrument (Changzhou) Co., Ltd.; LABOSPECT003 automatic biochemical analyzer, Hitachi, Japan; AniView100 multi-mode animal live imaging system, ANDOR; TDZ5-WS desktop multi-tube automatic balancing centrifuge, Hunan Keda Industrial Development Co., Ltd.; ME2002E electronic balance, Shimadzu Corporation, Japan; flow cytometer, BD; ASP200S fully automatic tissue dehydrator, ASP300S fully automatic tissue dehydrator, TP1020 fully automatic dehydrator, HI1210 slide spreader, HI1220 slide baking machine, RM2235 paraffin sectioner, EG1150H+C tissue embedding machine, AutoStainer XL automatic slide stainer + CV5030 automatic coverslipper, BX43 biological microscope + MD50 digital imaging system, CX31 biological microscope, Leica, Germany.

[0332] Experimental methods

[0333] The concentration of Luc-labeled fluorescence was 1×10 7Hepatocellular carcinoma (H22) cells at a concentration of 1 × 10 cells / mL were first inoculated into the peritoneal cavity of 8 male C57 mice. When the mice developed ascites, the ascites was aspirated aseptically, washed with HBSS buffer, centrifuged and the supernatant discarded, stained with trypan blue, and the ascites cells were counted under a microscope. The cell count was adjusted to 1 × 10 cells / mL with HBSS buffer. 13 Fifty-one male C57 mice were inoculated with 10 μL of cisplatin per mouse into the right liver region to create orthotopic tumor-bearing mice. One week later, liver tumor formation in the mice was assessed using a small animal in vivo imaging device. The mice were randomly divided into the following groups according to tumor size: model control group, cisplatin group (4 mg / kg), Kanglaite soft capsule group (1404 mg / kg), cisplatin + Kanglaite soft capsule group (4 + 1404 mg / kg), cisplatin + GLP-4 low-dose group (4 + 130 mg / kg), and cisplatin + GLP-4 high-dose group (4 + 1170 mg / kg), with six mice in each group. Six mice were also selected as the normal control group. The normal and model control groups were gavaged with purified water, while the chemotherapy group received an intraperitoneal injection of cisplatin. The remaining groups received the corresponding drug solutions at a volume of 20 mL / kg (gavage) or 10 mL / kg (intraperitoneal injection), respectively, once daily for 14 consecutive days. After the last administration, blood was collected from the orbit to detect blood WBC, RBC, liver and kidney function indicators (ALT, AST, BUN, CRE) and CD3 + / CD4 + 、CD3 + / CD8 + , the spleen, thymus, and tumor were weighed and the organ coefficient was calculated.

[0334] Dosage design

[0335] Ganoderma lucidum polysaccharide GLP-4 was designed based on previous experimental results. The low dose was 130 mg / kg and the high dose was 1170 mg / kg. The corresponding drugs were administered as shown in Table 18.

[0336] The proposed clinical dosage of Kanglaite Soft Capsules is 0.45g / capsule, 6 capsules / time, 4 times / day, which is 10.8g / day. The equivalent dose in mice based on body surface area is 10.8g / day*0.0026 / 0.02kg=1404mg / kg. This trial was conducted at the same multiple of the proposed clinical dosage.

[0337] Cisplatin dosage design basis: According to the clinical dosage of cisplatin, each person should not exceed 100 mg / m2 per day. 2 The tolerance of mice to cisplatin was evaluated, and a dose of 4 mg / kg was selected as the administration dose.

[0338] Table 18 Trial grouping and dosage design

[0339] Detection indicators

[0340] Efficacy indicators

[0341] Animal survival rate and general condition: The animals were weighed once a week and the death of animals was recorded.

[0342] Tumor volume detection: Small animal in vivo imaging technology was used to detect the changes in tumor volume in each group every week.

[0343] Hematological examination: blood routine (WBC, RBC) and biochemical (liver and kidney function) indexes were tested after the last dose.

[0344] Immune organs: Weigh the thymus, spleen, tumor, and liver, and calculate the organ coefficient. Organ coefficient (%) = organ weight / fasting body weight × 100%

[0345] CD4 + 、CD8 + Content detection: After the last administration, blood lymphocyte typing CD3 was detected by flow cytometry + / CD4 + , CD3 + / CD8 + content.

[0346] Data processing and statistical analysis

[0347] The significant figures of the experimental data were rounded off and statistical analysis was performed in accordance with the center's SOP. The statistical software used was SPSS. Normality and homogeneity of variance were tested using the Leven's test. If there was no statistical significance (P>0.05), one-way analysis of variance (ANOVA) was used for statistical analysis. If the ANOVA was statistically significant (P≤0.05), the LSD test (parametric method) was used for comparative analysis. If the variances were unequal (P≤0.05), the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P≤0.05), the Dunnett's test (nonparametric method) was used for comparative analysis. Statistical results were tested with an α=0.05 threshold, where P≤0.05 indicated statistical significance and P≤0.01 indicated that the difference being tested was highly significant.

[0348] Experimental results

[0349] General clinical observations and animal deaths

[0350] As shown in Table 19, mice were active before dosing, with normal movements and gait. Following dosing, activity decreased, and tumor growth affected their diet and water intake, leading to minimal weight gain. 2M01 in the model control group died on D12; 3M01 / 3M05 and 3M02 in the cisplatin group died on D9 and D10, respectively; 4M01 in the Kanglaite soft capsule group died on D9; 5M01 and 5M06 in the cisplatin + Kanglaite soft capsule group died on D8 and D11, respectively; and 8M02 in the cisplatin + low-dose GLP-4 group died on D8.

[0351] The mortality rates of each group were 0%, 16.7%, 50.0%, 16.7%, 16.7%, 16.7%, and 0%, respectively.

[0352] Table 19 Statistics of the number of surviving animals and mortality rates in each group

[0353] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on body weight in mice bearing H22 orthotopic hepatocellular carcinoma

[0354] As shown in Table 20, compared with the normal control group, the body weight of mice in the model control group was significantly reduced at week 2 after administration (P ≤ 0.01). Compared with the model control group, the body weight of mice in the cisplatin + GLP-4 low-dose group and the cisplatin + GLP-4 high-dose group was significantly reduced at week 1 and week 2 after administration (P ≤ 0.05 or P ≤ 0.01). The body weight of mice in the cisplatin group and the cisplatin + Kanglaite soft capsule group was significantly reduced at week 1 and week 2 after administration (P ≤ 0.05 or P ≤ 0.01). Compared with the cisplatin group, the body weight of mice in the Kanglaite soft capsule group was significantly increased at week 2 after administration (P ≤ 0.05 or P ≤ 0.01). Compared with the Kanglaite soft capsule group, the body weight of mice in the cisplatin combined with GLP-4 low-dose and high-dose groups was significantly reduced at week 1 and week 2 after administration (P ≤ 0.05 or P ≤ 0.01). Compared with the cisplatin + Kanglaite soft capsule group, the body weight of mice in the cisplatin combined with GLP-4 high-dose group was significantly reduced after 2 weeks of administration (P≤0.05 or P≤0.01).

[0355] Table 20 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on body weight of mice with H22 orthotopic liver cancer

[0356] Note: Compared with the normal control group, ++ P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01; compared with the cisplatin group, # P≤0.05, ## P≤0.01; compared with Kanglaite soft capsule group, & P≤0.05, && P≤0.01; compared with the cisplatin + Kanglaite soft capsule group, ★P≤0.05, ★★ P≤0.01.

[0357] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 orthotopic hepatocellular carcinoma in mice

[0358] As shown in Figures 35, 36, and Table 21, compared with the normal control group, the tumors in the model control group mice increased significantly (P ≤ 0.01); compared with the model control group, the tumors in mice W1 and W2 administered with Kanglaite soft capsules were significantly reduced (P ≤ 0.05 or P ≤ 0.01); the tumors in mice W2 administered with cisplatin combined with low- and high-dose GLP-4 groups, the cisplatin group, and the cisplatin combined with Kanglaite soft capsules group were significantly reduced (P ≤ 0.05 or P ≤ 0.01). Compared with the cisplatin group, there were no significant differences among the treatment groups. Compared with the Kanglaite soft capsule group, the tumors in mice W1 administered with the high-dose cisplatin combined with GLP-4 group and the Kanglaite soft capsule group were significantly increased (P ≤ 0.05 or P ≤ 0.01). Compared with the cisplatin + Kanglaite soft capsule group, there were no statistical differences among the groups.

[0359] As shown in Figure 35, the corresponding groups are: A: normal group, B: model control group, C: cisplatin group, D: Kanglaite soft capsule group, E: cisplatin + Kanglaite soft capsule group, H: cisplatin + GLP-4 low-dose group, I: cisplatin + GLP-4 high-dose group.

[0360] As shown in Figure 36, the corresponding groups are: 1: normal group, 2: model control group, 3: cisplatin group, 4: Kanglaite soft capsule group, 5: cisplatin + Kanglaite soft capsule group, 8: cisplatin + GLP-4 low-dose group, 9: cisplatin + GLP-4 high-dose group.

[0361] Table 21 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 orthotopic liver cancer in mice

[0362] Note: Compared with the normal control group, ++ P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01; compared with the cisplatin group, # P≤0.05, ## P≤0.01; compared with Kanglaite soft capsule group, & P≤0.05, && P≤0.01; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0363] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on organ coefficients and tumor growth inhibition rate in mice with H22 orthotopic hepatocellular carcinoma

[0364] As shown in Table 22, compared with the normal control group, the organ coefficients of the spleen and liver tissues in the model control group were significantly increased (P ≤ 0.05 or P ≤ 0.01). Compared with the model control group, the thymus and spleen coefficients of mice in the low- and high-dose cisplatin combined with GLP-4 groups were significantly decreased (P ≤ 0.05 or P ≤ 0.01), and the thymus and spleen coefficients of mice in the cisplatin group and the cisplatin + Kanglaite soft capsule group were significantly decreased (P ≤ 0.05). Compared with the cisplatin group, there were no significant differences among the groups. Compared with the Kanglaite soft capsule group, the thymus and spleen coefficients of mice in the low-dose cisplatin combined with GLP-4 group were significantly decreased (P ≤ 0.05 or P ≤ 0.01), and the spleen coefficients of mice in the high-dose cisplatin combined with GLP-4 and Kanglaite soft capsule groups were significantly decreased (P ≤ 0.05 or P ≤ 0.01).

[0365] Table 22 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on organ coefficients of H22 in situ liver cancer mice

[0366] Note: Compared with the normal control group, + P≤0.05, ++ P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01; compared with the cisplatin group, # P≤0.05; compared with Kanglaite soft capsule group, & P≤0.05, && P≤0.01.

[0367] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on blood biochemical parameters in mice with H22 orthotopic hepatocellular carcinoma

[0368] As shown in Table 23, compared with the normal control group, the blood AST and CRE of mice in the model control group were significantly increased (P ≤ 0.01). Compared with the model control group, the blood WBC of mice in the cisplatin combined with Kanglaite soft capsule group was significantly decreased (P ≤ 0.05), the blood WBC and BUN of mice in the cisplatin combined with GLP-4 low-dose group were significantly decreased (P ≤ 0.05 or P ≤ 0.01), and the blood WBC and RBC of mice in the cisplatin combined with GLP-4 high-dose group were significantly decreased (P ≤ 0.05 or P ≤ 0.01). Compared with the cisplatin group, the blood BUN of mice in the cisplatin combined with GLP-4 low-dose group was significantly decreased (P ≤ 0.05). Compared with the Kanglaite soft capsule group, the blood RBC of mice in the cisplatin combined with GLP-4 low-dose group was significantly decreased (P ≤ 0.05 or P ≤ 0.01), and the blood CRE of mice in the cisplatin combined with GLP-4 high-dose group was significantly increased (P ≤ 0.05). There was no statistical significance in the other groups.

[0369] Table 23 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on blood biochemical indicators in mice with H22 in situ liver cancer

[0370] Note: Compared with the normal control group, + P≤0.05, ++ P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01; compared with the cisplatin group, # P≤0.05, ## P≤0.01; compared with Kanglaite soft capsule group, & P≤0.05, && P≤0.01; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0371] As shown in Table 24, compared with the normal control group, the blood CD3+ / CD4+ and CD3+ / CD8+ of mice in the model control group showed an upward trend, but there was no statistical significance. + / CD8 + The CD3 + / CD4 + 、CD3 + / CD8 + Compared with the cisplatin group, the CD3 + / CD4 + 、CD3 + / CD8 + Compared with the Kanglaite soft capsule group, the CD3 + / CD4 + 、CD3 + / CD8 + The levels of CD3 in the blood of mice in the high-dose cisplatin combined with GLP-4 group were significantly increased (P≤0.05). + / CD4 + Compared with the cisplatin + Kanglaite soft capsule group, the CD3 + / CD4 + All of them were significantly increased (P≤0.05).

[0372] Table 24 Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on blood CD3 in H22 orthotopic liver cancer mice + / CD4 + 、CD3 + / CD8+ Impact

[0373] Note: Compared with the model control group, *P≤0.05; compared with the cisplatin group, # P≤0.05; compared with Kanglaite soft capsule group, & P≤0.05; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05.

[0374] As shown in Figures 37-40, mice in the model control group showed extensive liver cancer cell infiltration and necrosis, an increase in the number of sinusoidal cells, and inflammatory cell infiltration. Extramedullary hematopoiesis in the spleen increased significantly, with diffuse red pulp. The stomach showed liver cancer cell infiltration, lesions in the serosal membrane, and extensive atrophy of the gastric mucosa. Following administration of GLP-4, a polysaccharide derived from Ganoderma lucidum, combined with cisplatin, liver cancer cell infiltration and necrosis in all groups were reduced, extramedullary hematopoiesis in the spleen increased, and no significant lesions were observed in the stomach or kidneys.

[0375] As shown in Figures 37-40, the corresponding groups are: A: normal group, B: model control group, C: cisplatin group, D: Kanglaite soft capsule group, E: cisplatin + Kanglaite soft capsule group, F: cisplatin + GLP-4 low dose group, G: cisplatin + GLP-4 high dose group

[0376] in conclusion

[0377] Ganoderma lucidum polysaccharide GLP-4 combined with cisplatin can significantly inhibit the growth of tumors in H22 orthotopic liver cancer mice and has a significant synergistic effect.

[0378] Discussion and Summary

[0379] Liver cancer is a malignant tumor with an extremely high mortality rate. Currently, surgical resection combined with chemotherapy and interventional treatment, as well as radiotherapy, only serve to alleviate symptoms, making a complete cure extremely difficult. Liver cancer is not sensitive to chemotherapy, especially in patients with advanced liver cancer. To date, there is no reliable evidence that systemic chemotherapy can improve overall survival in patients with advanced liver cancer.

[0380] The results of this experiment showed that the tumor volume of the mice in the model control group increased significantly, the spleen index and liver index increased significantly, the number of red blood cells in the blood increased significantly, the number of white blood cells decreased significantly, and the liver and kidney functions were significantly abnormal, indicating that the lymphatic system function of the mice in the model control group decreased during the tumorigenesis process. After the last administration, GLP-4, a polysaccharide of Ganoderma lucidum, combined with cisplatin, significantly inhibited the growth of tumors in mice, significantly reduced the thymus index and spleen index, significantly reduced the number of red blood cells in the blood of mice, significantly increased the number of white blood cells, significantly reduced the content of kidney function indicators, and significantly reduced liver function indicators. CD4 + T cells and CD8 + T cells mediate tumor immune responses, including CD8+ T cells are the main effector cells of tumor immunity. The results showed that GLP-4 combined with cisplatin in the blood of mice with Ganoderma lucidum polysaccharide had a significant effect on the expression of CD3 + / CD4 + 、CD3 + / CD8 + The content of GLP-4 in the blood was significantly increased, indicating that the combination of GLP-4 and cisplatin can protect immune organs and enhance the body's autoimmune function, thereby reducing toxicity and improving anti-tumor effects. Histopathological results also showed that GLP-4 can enhance the body's immunity. In addition, compared with Kanglaite soft capsules combined with cisplatin, the combination of GLP-4 and cisplatin significantly reduced the tumor, spleen, and thymus coefficients. These experimental results show that the anti-tumor effect of GLP-4 and cisplatin is stronger than that of Kanglaite soft capsules combined with cisplatin.

[0381] In summary, Ganoderma lucidum polysaccharide GLP-4 combined with cisplatin can significantly inhibit the growth of tumors in H22 orthotopic liver cancer mice and has a significant synergistic effect.

[0382] Co-localization detection experiment and data of Ganoderma lucidum polysaccharide GLP-4 and RAW264.7 macrophage lysosomes

[0383] Purpose of the experiment:

[0384] The co-localization detection of Ganoderma lucidum polysaccharide GLP-4 and RAW264.7 macrophage lysosomes further elucidated the mechanism of drug action and provided experimental basis for its clinical research.

[0385] Experimental Materials

[0386] Test sample

[0387] Ganoderma lucidum polysaccharide GLP-4; batch number: OLMJT202108(2-4), provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0388] cell lines

[0389] RAW264.7 macrophage cell line was purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0390] Main reagents

[0391] PBS was prepared by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.; DMEM medium was a product of Gibco; Cell Navigator lysosome staining kit was a product of Beijing Solebaugh Technology Co., Ltd.; DAPI dye was a product of Tianjin Bioma Technology Co., Ltd.; HBSS buffer was a product of Gibco.

[0392] Main instruments

[0393] 20μL, 200μL, and 1mL pipettes were products of Mettler-Toledo Co., Ltd.; Luna-Ⅱ cell counter was a product of Nanjing Hengqiao Instrument Co., Ltd.; laser confocal microscope was a product of Leica.

[0394] Experimental methods:

[0395] RAW264.7 macrophages were cultured in DMEM supplemented with 10% serum at 37°C in a 5% CO2 incubator until the logarithmic growth phase. 150,000 cells were added to a six-well plate and cultured for 1-2 days. Once the cell density reached above 50%, fluorescently labeled GLP-4 was added and incubated for 24 hours. Lysosomal stain and DAPI dye were then added, and images were captured using a laser confocal microscope at 488 nm, 550 nm, and UV light.

[0396] Test results

[0397] As shown in Figures 41-45, after incubation of Ganoderma lucidum polysaccharide GLP-4 with RAW264.7 macrophages for 24 hours, most cells contained the drug, indicating that GLP-4 can be phagocytosed by macrophages; the co-localization image of fluorescently labeled GLP-4 and lysosomes shows that the drug enters the macrophages through the endocytic pathway of the lysosomes; in Figure 44, the dead cells labeled with DAPI dye contain fluorescently labeled GLP-4, but there are no lysosomes in the cells, indicating that the lysosomes of macrophages cannot fully digest the drug. Therefore, GLP-4 can be presented by macrophages to other immune cells, successfully activating the immune system and achieving the effect of enhancing immunity.

[0398] Figure 41 shows confocal images of a blank control group of RAW264.7 macrophages at 40x magnification. A: A merged image of a brightfield image and a DAPI-stained image; B: An image in the FITC fluorescence channel; C: An image of lysosomes labeled with a lysosomal fluorescent dye; D: A merged image of C and the brightfield image.

[0399] Figure 42 shows confocal images of RAW264.7 macrophages incubated with 0.25 mg / mL GLP-4 for 24 hours at 40x magnification. A: A merged image of the brightfield image and the DAPI-stained image; B: An image of FITC-labeled GLP-4; C: An image of lysosomes labeled with a lysosomal fluorescent dye; D: A merged image of panels B and C, with the arrow indicating colocalization of GLP-4 and lysosomes.

[0400] Figure 43 shows confocal images of RAW264.7 macrophages incubated with 0.25 mg / mL GLP-4 for 24 hours, taken under a 63x oil microscope. A: A merged image of the brightfield image and the DAPI-stained image; B: An image of FITC-labeled GLP-4; C: An image of lysosomes labeled with a lysosomal fluorescent dye; D: A merged image of panels B and C; the arrow indicates colocalization of GLP-4 and lysosomes.

[0401] Figure 44 shows confocal images at 40x magnification after 24 hours of incubation of RAW264.7 macrophages with 0.5 mg / mL GLP-4. A: A merged image of a brightfield image and a DAPI-stained image; B: An image of FITC-labeled GLP-4; C: An image of lysosomes labeled with a lysosomal fluorescent dye; D: A merged image of panels B and C; the arrow indicates colocalization of GLP-4 and lysosomes.

[0402] Figure 45 shows confocal images of RAW264.7 macrophages incubated with 0.5 mg / mL GLP-4 for 24 hours, taken under a 63x oil microscope. A: A merged image of the brightfield and DAPI-stained images; B: An image of FITC-labeled GLP-4; C: An image of lysosomes labeled with a lysosomal fluorescent dye; D: A merged image of panels B and C; the arrow indicates colocalization of GLP-4 and lysosomes.

[0403] in conclusion

[0404] Ganoderma lucidum polysaccharide GLP-4 can be phagocytosed by RAW264.7 macrophages and activate the body's immunity.

[0405] Experimental study and data on the killing effect of Ganoderma lucidum polysaccharide GLP-4 combined with cisplatin on H22 liver cancer cells

[0406] Purpose of the experiment:

[0407] To explore the killing effect of Ganoderma lucidum polysaccharide GLP-4 on H22 liver cancer cells and provide experimental basis for its clinical research.

[0408] Experimental Materials

[0409] Test sample

[0410] Ganoderma lucidum polysaccharide GLP-4; batch number: OLMJT202108(2-4), provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0411] Positive control

[0412] Cisplatin, batch number: E2128081, product of Shanghai Aladdin Biochemical Technology Co., Ltd.

[0413] cell lines

[0414] H22 hepatocellular carcinoma cell line was purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0415] Main reagents

[0416] PBS was prepared by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.; 0.25% trypsin was a product of Gibco; 1640 culture medium was a product of Gibco.

[0417] Main instruments

[0418] 20μL, 200μL, and 1mL pipettes were products of Mettler-Toledo Co., Ltd.; Luna-Ⅱ cell counter was a product of Nanjing Hengqiao Instrument Co., Ltd.; laser confocal microscope was a product of Leica.

[0419] Experimental methods:

[0420] H22 cells were cultured in 1640 culture medium containing 10% serum in a 37°C, 5% CO2 incubator until the logarithmic growth phase. The culture medium was discarded from the cells in the culture flask, washed with PBS, and digested with 0.125% trypsin. After digestion, the culture medium was used to terminate the digestion. The cells were transferred to EP tubes and counted using a cell counter. After counting, 40,000 cells / well were aspirated and added to a 12-well plate and cultured in a 37°C, 5% CO2 incubator. After 24 hours of cell attachment, the drugs were added. 48 hours after drug addition, the cells were digested with trypsin and washed with PBS. After washing, the cells were counted. The corresponding volume of staining solution was added according to the number of cells and incubated in the incubator for 30 minutes. After incubation, the cells were placed in an ice box to terminate the staining and maintain cell activity. The cells were photographed using a laser confocal microscope.

[0421] Dosage design

[0422] According to the results of previous studies, the dosage concentration of Ganoderma lucidum polysaccharide GLP-4 was set to 2 mg / mL, and the dosage concentration of cisplatin (DDP) was set to 5 ug / mL.

[0423] Test results

[0424] As shown in Table 25 and Figure 46, GLP-4 can induce necrosis in H22 cells, and when used in combination with cisplatin, it can reduce the cell apoptosis and necrosis caused by cisplatin, and can reduce the direct toxicity of cisplatin.

[0425] Table 25 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with cisplatin on necrosis and apoptosis of H22 cells

[0426] As shown in Figure 46, this is the effect of GLP-4, a polysaccharide of Ganoderma lucidum, combined with cisplatin on the necrosis rate and apoptosis rate of H22 cells.

[0427] in conclusion

[0428] Ganoderma lucidum polysaccharide GLP-4 can induce necrosis of H22 cells, and combined with cisplatin can reduce cell apoptosis and necrosis caused by cisplatin, and can reduce the direct toxicity of cisplatin.

[0429] Experimental study and data on the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on 4T1 breast cancer-bearing mice

[0430] Purpose of the experiment

[0431] BALB / c mice were subcutaneously inoculated with 4T1 breast cancer cells on their left shoulder to prepare tumor-bearing mice. The effect of Ganoderma lucidum polysaccharide GLP-4 on breast cancer tumor-bearing mice was studied to provide an experimental basis for its clinical research.

[0432] Experimental Materials

[0433] Test sample

[0434] Ganoderma lucidum polysaccharide GLP-4, batch number: OLM2019071617; provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0435] Positive control

[0436] Cisplatin, batch number: E2128081, product of Shanghai Aladdin Biochemical Technology Co., Ltd.

[0437] experimental animals

[0438] Sixty-five SPF female BALB / c mice, weighing 14–16 g, were provided by the Guangdong Medical Laboratory Animal Center. The laboratory animal production license number is SCXK (Guangdong) 2022-0002, and the laboratory animal quality certificate number is 44007200100339.

[0439] Main reagents

[0440] PBS buffer was prepared by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.; fetal bovine serum was a product of Zhejiang Tianhang Biotechnology Co., Ltd.; 1640 culture medium was a product of Gibco; and 0.25% trypsin was a product of Gibco.

[0441] Main instruments

[0442] Vernier caliper, product of Shanghai Tool Factory Co., Ltd.; I-2000 balance, product of Dongguan Nancheng Changxie Electronic Products Factory; ophthalmic scissors and tweezers, product of Shanghai Jinzhong Medical Instrument Co., Ltd.; CCL-170B-8 carbon dioxide incubator, product of Singapore ESCO Company; Luna-Ⅱ cell counter, product of Nanjing Hengqiao Instrument Co., Ltd.

[0443] Experimental methods

[0444] A solid tumor model was established by subcutaneously injecting a suspension of 4T1 breast cancer cells into the left shoulder of 55 healthy female BALB / c mice. When the average tumor volume reached approximately 240 mm³, the mice were randomly divided into groups based on tumor volume and given the corresponding drug or vehicle by gavage or intraperitoneal injection for 25 consecutive days. Tumor diameters were measured every three days to calculate tumor volume, and mice were weighed every three days. At the end of the experiment, tumors, spleens, and thymuses were weighed, and the tumor index, spleen index, and thymus index were calculated.

[0445] Dosage design

[0446] Ganoderma lucidum polysaccharide GLP-4 was designed based on previous experimental results. The low dose was 130 mg / kg and the high dose was 1170 mg / kg. The corresponding drugs were administered as shown in Table 26.

[0447] Cisplatin dosage design basis: According to the clinical dosage of cisplatin, each person should not exceed 100 mg / m2 per day. 2 The tolerance of mice to cisplatin was evaluated, and a dose of 4 mg / kg was selected as the administration dose.

[0448] Table 26 Trial grouping and dosage design

[0449] Detection indicators

[0450] Efficacy indicators

[0451] Relative tumor inhibition rate

[0452] Relative tumor inhibition rate (%) = (1-T RTV / C RTV )×100%. Among them, T RTV is the relative tumor volume of the experimental group, C RTV is the relative tumor volume of the model control group. t / V0,V t =V0 is the tumor volume of mice on day t of drug administration, and V0 is the tumor volume of mice at the time of grouping. Evaluation criteria: Relative tumor inhibition rate ≥ 40%, and P < 0.05 after statistical analysis is considered an effective inhibition level.

[0453] Tumor growth inhibition rate

[0454] Tumor growth inhibition rate (%) = (1-T / C) × 100%. Where T represents the average tumor weight of the treatment group and C represents the average tumor weight of the model control group. Evaluation criteria: Tumor growth inhibition rate ≥ 40% and statistical analysis P < 0.05 are considered effective inhibition levels.

[0455] Spleen and thymus organ coefficients: After the last administration, the spleen, thymus, and tumor were weighed and the organ coefficients were calculated.

[0456] Data processing and statistical analysis

[0457] SPSS 17.0 was used for statistical analysis. The level of statistical significance was set at P ≤ 0.05. Measurement data were expressed as mean ± standard deviation (x ± s). Leven's test was used to test normality and homogeneity of variance. If normality and homogeneity of variance were met (P > 0.05), one-way analysis of variance (ANOVA) and LSD test were used for statistical analysis. If normality and homogeneity of variance were not met (P < 0.05), Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P < 0.05), Dunnett's Test (non-parametric method) was used for comparative analysis. Statistical differences and biological significance were considered in the evaluation.

[0458] Experimental results

[0459] Animal deaths

[0460] As shown in Table 27, the mortality rate of mice in the model control group was 60%, and the mortality rate of mice in the other groups was 0.

[0461] Table 27 Statistics of surviving animals and mortality rates in each group

[0462] Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on body weight in 4T1 breast cancer-bearing mice

[0463] As shown in Table 28, compared with the model control group, the body weight of mice in the cisplatin group, the low-dose cisplatin + GLP-4 group, and the high-dose cisplatin + GLP-4 group decreased significantly from D3 to D22. Compared with the cisplatin group, the body weight of mice in the high-dose cisplatin + GLP-4 group decreased significantly on D19 and D22.

[0464] Table 28 Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the body weight of 4T1 breast cancer tumor-bearing mice

[0465] Note: Compared with the model control group, * P<0.05; compared with the cisplatin group, # P<0.05.

[0466] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on tumor volume in 4T1 breast cancer-bearing mice

[0467] As shown in Table 29, compared with the model control group, the tumor volumes of the cisplatin group, the cisplatin + GLP-4 low-dose group, and the cisplatin + GLP-4 high-dose group were significantly reduced from D3 to D22.

[0468] Compared with the cisplatin group, the tumor volume at D12 in the low-dose cisplatin + GLP-4 group was significantly reduced, and the tumor volume at D19 and D22 in the high-dose cisplatin + GLP-4 group was significantly reduced.

[0469] Table 29 Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on tumor volume in 4T1 breast cancer-bearing mice

[0470] Note: Compared with the model control group, * P<0.05; compared with the cisplatin group, # P<0.05.

[0471] As shown in Table 30, compared with the model control group, the relative tumor inhibition rate of the cisplatin group was above 40% from D6 to D25, with a maximum of 56.9%. The relative tumor inhibition rate of the cisplatin + GLP-4 low-dose group was above 50% from D6 to D25, with a maximum of 75.2%. The relative tumor inhibition rate of the cisplatin + GLP-4 high-dose group was above 55% from D6 to D18, with a maximum of 74.0%.

[0472] Compared with the cisplatin group, the relative tumor inhibition rate of the low-dose cisplatin + GLP-4 group was 22% to 44% from D6 to D22 (P<0.05), and the relative tumor inhibition rate of the high-dose cisplatin + GLP-4 group was 24% to 50% from D6 to D25 (P<0.05).

[0473] Table 30 Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the relative tumor inhibition rate of 4T1 breast cancer-bearing mice (compared with the model control group)

[0474] Note: Compared with the model control group, * P<0.05.

[0475] Table 31 Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the relative tumor inhibition rate of 4T1 breast cancer-bearing mice (compared with the cisplatin group)

[0476] Note: Compared with cisplatin group, # P<0.05.

[0477] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on organ coefficients and tumor growth inhibition rate in 4T1 breast cancer-bearing mice

[0478] As shown in Table 32, compared with the model group, the tumor index, spleen index, and thymus index in the cisplatin combined with GLP-4 group were significantly reduced. Compared with the cisplatin group, the tumor index in the high-dose cisplatin + GLP-4 group was significantly reduced, and the spleen index in the cisplatin combined with GLP-4 group was significantly reduced.

[0479] Compared with the model control group, the tumor growth inhibition rates in the cisplatin group were 29.6%, 49.5% in the low-dose cisplatin + GLP-4 group, and 60.1% in the high-dose cisplatin + GLP-4 group. Compared with the cisplatin group, the tumor growth inhibition rates in the low-dose cisplatin + GLP-4 group and high-dose cisplatin + GLP-4 group were 28.2% and 43.3%, respectively.

[0480] Table 32 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on organ index and tumor growth inhibition rate in 4T1 breast cancer-bearing mice

[0481] Note: Compared with the model control group, * P<0.05; compared with the cisplatin group, # P<0.05.

[0482] As shown in Figures 47-50, these are pictures of tumor-bearing mice, and the corresponding groups are: Figure 47: Model control group, Figure 48: Cisplatin group, Figure 49: Cisplatin + GLP-4 low-dose group, Figure 50: Cisplatin + GLP-4 high-dose group

[0483] Figures 51-54 show tumor images of tumor-bearing mice, and the corresponding groups are: Figure 51: Model control group, Figure 52: Cisplatin group, Figure 53: Cisplatin + GLP-4 low-dose group, Figure 54: Cisplatin + GLP-4 high-dose group

[0484] in conclusion

[0485] Ganoderma lucidum polysaccharide GLP-4 combined with cisplatin can significantly inhibit the growth of tumors in 4T1 breast cancer-bearing mice and has a synergistic effect.

[0486] Bleomycin-induced pulmonary fibrosis animal model efficacy experiments and data

[0487] Purpose of the experiment

[0488] This study investigated the therapeutic effect of Ganoderma lucidum polysaccharide GLP-4 on pulmonary fibrosis using a bleomycin-sensitized mouse pulmonary fibrosis model, providing an experimental basis for its clinical research.

[0489] Experimental Materials

[0490] Test sample

[0491] Ganoderma lucidum polysaccharide GLP-4, batch number: OLMJT202208 (20, 23, 24), was provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0492] Positive control

[0493] Pirfenidone capsules, batch number: 20220606, were provided by Boji Pharmaceutical Technology Co., Ltd.

[0494] experimental animals

[0495] Fifty SPF-grade C57BL / 6J mice, weighing 21.1–25.0 g, were purchased from Guangdong Weitonglihua Laboratory Animal Technology Co., Ltd. (Laboratory Animal Production License No. SCXK(Yue)2020-0063). Laboratory Animal Quality Certificate No. 44829700008922.

[0496] Main reagents

[0497] Bleomycin, batch number: C13661083, product of Maclean.

[0498] Main instruments

[0499] TW423L electronic balance, product of Shimadzu Corporation of Japan; YP2001 electronic balance, product of Shanghai Jinping Company; JJ124BC electronic balance, product of Changshu Shuangjie Company; EMKA-WBP animal lung function testing system, product of EMKA Company.

[0500] Experimental methods

[0501] Fifty SPF male C57BL / 6J mice were randomly divided by body weight into three groups: a normal control group, a model control group, a positive control group, a low-dose GLP-4 group, and a high-dose GLP-4 group. Ten animals were selected as the normal control group, and the remaining animals were used for modeling, with 10 mice in each group. Modeling was initiated by intratracheal administration of bleomycin (5 mg / kg, 0.1 mL / mouse) once. On day 7 of modeling, the test drug, positive drug, or vehicle were administered orally at 10 mL / kg once daily for 21 consecutive days. During the experiment, the mice in each group were observed daily for diet, activity, breathing, and reflexes. Lung function was measured before and approximately 24 hours after the last dose. After pulmonary function testing, the mice were euthanized and pathological specimens were collected.

[0502] Dosage design

[0503] The proposed clinical dosage of Ganoderma lucidum polysaccharide GLP-4 is 1.0g per day. Based on an adult weight of 60kg and a mouse weight of 20g, and a body surface area conversion coefficient of 0.0026 between humans and mice, the clinical equivalent dose for mice is approximately 187mg / kg. The designed low (approximate clinical equivalent dose) and high doses of GLP-4 are 200mg / kg and 600mg / kg, respectively.

[0504] The positive drug pirfenidone was calculated based on a stable daily adult dose of 1200 mg. The clinical equivalent dose in mice was 225 mg / kg, which was set as the positive control dose, as shown in Table 33.

[0505] Table 33 Dosage design and administration schedule

[0506] Note: The dose conversion formula is: logS = 0.8762 + 0.698logW (S: body surface area, unit is cm 2 ; W: body weight, in g). Human body weight is calculated as 60 kg, and mouse body weight is calculated as 20 g / mouse.

[0507] Detection indicators

[0508] General behavioral observations: During the experiment, the diet, activity, breathing and reaction ability of each group of animals were observed every day.

[0509] Pulmonary function: Before administration and approximately 24 hours after the last administration, the EMKA WBP animal pulmonary function testing system was used to test the lung function of mice, which can include the duration of inspiration (expiration), maximum inspiration (expiration), tidal volume, expiratory volume, relaxation time, minute ventilation, respiratory rate, end-inspiratory (end-expiratory) space, bronchial contraction parameters, 50% expiratory flow, alveolar ventilation, respiratory rate, etc.

[0510] Lung tissue pathology: After the pulmonary function test, the mice were killed, the abdomen was opened, and the whole body was perfused with normal saline through the portal vein. The chest was opened, and the lungs were fixed with 10% formaldehyde solution for 5 minutes, then the trachea was ligated, and the lungs were removed and fixed with formalin solution for 48 hours. HE staining was performed to observe the pathological conditions.

[0511] Data processing and statistical analysis

[0512] The experimental measurement data were summarized in Excel and expressed as mean ± standard deviation. SPSS 28.0 statistical software was used for analysis. One-way analysis of variance (ANOVA) was used for variance homogeneity test. When the variance was homogeneous (P ≥ 0.05), the LSD test was used for inter-group comparison. When the variance was unequal (P < 0.05), the Dunnett's T3 test was used for inter-group comparison. P < 0.05 was considered statistically significant.

[0513] Experimental results

[0514] General behavioral observations

[0515] During the trial, compared with the normal control group, the mice in the model control group had poor appetite, decreased activity, accelerated breathing, weakened reflexes, and dull fur. Compared with the model control group, the mice in the positive control group, low-dose GLP-4 group, and high-dose GLP-4 group showed significant improvements in appetite, activity, breathing, reflexes, and fur.

[0516] Pulmonary function tests

[0517] Before drug administration (one week after modeling), lung function tests were performed as shown in Tables 34 and 35. Compared with the normal control group, the model control group showed significantly increased inspiratory duration (Ti), maximum inspiratory volume (PIF), relaxation time (RT), end-inspiratory interval (EIP), and respiratory rate (n), while minute ventilation (MV) and bronchial contraction parameter (Penh) were significantly decreased (p < 0.01). In the positive control group, the low- and high-dose GLP-4 groups showed similar indices to those in the model control group. These results suggest that lung function abnormalities were present in all modeling groups, indicating successful modeling. Statistically significant differences in maximum expiratory flow (PEF), MV, 50% expiratory flow (EF50), and n were observed in the positive control group compared with the model control group. These differences are likely due to individual animal differences and measurement error and are not clinically significant.

[0518] After 21 days of administration, lung function tests were performed as shown in Tables 36 and 37. Compared with the normal control group, the Ti, maximum expiratory flow (PEF), tidal volume (TV), expiratory volume (EV), MV, Penh, and 50% expiratory flow (EF50) of mice in the model control group were significantly decreased (p < 0.01 or 0.05). Compared with the model control group, the PIF, PEF, TV, EV, MV, EF50, and AV of mice in the positive control group and the low- and high-dose GLP-4 groups were significantly increased (p < 0.01 or 0.05). Ti also increased to a certain extent, with statistically significant differences between the positive control group and the low-dose GLP-4 group (p < 0.05). Compared with the positive control group, no statistical differences were observed in the drug-treated groups.

[0519] Table 34 Pulmonary function test results Table 1

[0520] Note: Compared with the normal control group, #p<0.05, ##p<0.01; compared with the model control group, *p<0.05, **p<0.01; compared with the positive drug control group, &p<0.05, &&p<0.01. Inspiratory duration (Ti), expiratory duration (Te), peak inspiratory flow (PIF), peak expiratory flow (PEF), tidal volume (TV), expiratory volume (EV), and relaxation time (RT).

[0521] Table 35 Pulmonary function test results Table 2

[0522] Note: #p<0.05, ##p<0.01 compared with the normal control group; *p<0.05, **p<0.01 compared with the model control group; &p<0.05, &&p<0.01 compared with the positive drug control group. Minute ventilation (MV), end-inspiratory interval (EIP), end-expiratory interval (EEP), bronchoconstriction parameter (Penh), 50% expiratory flow (EF50), alveolar ventilation (AV), and respiratory rate (n).

[0523] Table 36 Pulmonary function test results Table 3

[0524] Note: Compared with the normal control group, #p<0.05, ##p<0.01; compared with the model control group, *p<0.05, **p<0.01; compared with the positive drug control group, &p<0.05, &&p<0.01. Inspiratory duration (Ti), expiratory duration (Te), peak inspiratory flow (PIF), peak expiratory flow (PEF), tidal volume (TV), expiratory volume (EV), and relaxation time (RT).

[0525] Table 37 Pulmonary function test results Table 4

[0526] Note: #p<0.05, ##p<0.01 compared with the normal control group; *p<0.05, **p<0.01 compared with the model control group; &p<0.05, &&p<0.01 compared with the positive drug control group. Minute ventilation (MV), end-inspiratory interval (EIP), end-expiratory interval (EEP), bronchoconstriction parameter (Penh), 50% expiratory flow (EF50), alveolar ventilation (AV), and respiratory rate (n).

[0527] Pathological examination

[0528] The pathological examination results are shown in Table 38, which show that compared with the normal control group, the lung pathology scores of the model control group were significantly increased (p<0.01); compared with the model control group, the lung pathology scores of the GLP-4 low-dose group and the GLP-4 high-dose group were significantly decreased (p<0.01 or 0.05).

[0529] Table 38 Summary of pathological scores of each group

[0530] Note: Compared with the normal control group #p<0.05, ##p<0.01; compared with the model control group *p<0.05, **p<0.01; compared with the positive drug control group & p<0.05, && p<0.01.

[0531] As shown in Figures 55-59, the alveolar structure of mice in the normal control group was normal, with no inflammatory cell infiltration and no fibrosis. Mice in the model control group had lung tissue damage (40-60%), inflammatory cell infiltration within the lung tissue (40-60%), and fibrosis around blood vessels and in the lung tissue (40-60%). Mice in the positive control group had lung tissue damage (1-20%), inflammatory cell infiltration within the lung tissue (1-20%), and no fibrosis. Mice in the low-dose GLP-4 group had normal alveolar structure, inflammatory cell infiltration within the lung tissue (1-20%), and no fibrosis around blood vessels and in the lung tissue. Mice in the high-dose GLP-4 group had lung tissue damage (1-20%), inflammatory cell infiltration within the lung tissue (1-20%), and no fibrosis around blood vessels and in the lung tissue.

[0532] In Figures 55-59, the control groups are marked as follows: Figure 55: normal control group; Figure 56: model control group; Figure 57: positive control group; Figure 58: low-dose GLP-4 group; Figure 59: high-dose GLP-4 group

[0533] in conclusion

[0534] The low-dose group of Ganoderma lucidum polysaccharide GLP-4 significantly improved the appetite, activity, respiration, reaction ability, and coat condition of the model animals, significantly increased the Ti, PIF, PEF, TV, EV, MV, EF50, and AV of the model animals, improved lung tissue pathology, and had an improvement effect on alveolar tissue damage, inflammatory cell infiltration, and fibrous tissue hyperplasia; the high-dose group of Ganoderma lucidum polysaccharide GLP-4 improved the appetite, activity, respiration, reaction ability, and coat condition of the model animals, significantly increased the PIF, PEF, TV, EV, MV, EF50, and AV of the model animals, improved lung tissue pathology, and had an improvement effect on alveolar tissue damage, inflammatory cell infiltration, and fibrous tissue hyperplasia.

[0535] In summary, under the conditions of this experiment, Ganoderma lucidum polysaccharide GLP-4 can have a therapeutic effect on pulmonary fibrosis by improving the general behavioral status, lung function, lung tissue lesions, etc. of asthma model mice.

[0536] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Ganoderma lucidum polysaccharide GLP-4, characterized in that: The molecular structure of the Ganoderma lucidum polysaccharide GLP-4 Molecular formula: (C 162 H 270 O 135 ) n , where n=10-15.

2. The Ganoderma lucidum polysaccharide GLP-4 according to claim 1, characterized in that: n is 10, 11, 12, 13, 14 or 15.

3. A method for extracting GLP-4 from Ganoderma lucidum polysaccharide, characterized in that: The method comprises the following steps: S1, removing dust from the ganoderma lucidum, drying it, and then crushing it to make ganoderma lucidum powder; S2, placing the crushed ganoderma lucidum in a sealed container, mixing with water and heating, and fully blending the ganoderma lucidum powder and water into a medicinal juice solution under high temperature and high pressure; S3, using centrifugal technology to separate the medicinal juice solution to obtain a concentrated solution with medicinal ingredients and medicinal residues; S4, mixing the separated medicinal residues with the NaOH solution according to a preset ratio, soaking them, and then adding HCl to neutralize them; S5, concentrating and desalting the mixed solution by membrane concentration technology to remove the NaCl component in the mixed solution to obtain a concentrated solution with medicinal ingredients; S6. The concentrated solution containing the medicinal ingredients is freeze-dried and prepared into a certain concentration, and then separated by column chromatography for multiple times to obtain Ganoderma lucidum polysaccharide GLP-4 containing the medicinal ingredients.

4. The method for extracting GLP-4 from Ganoderma lucidum polysaccharide according to claim 3, characterized in that: In step S2, the ganoderma lucidum powder and water in the sealed container are mixed and stirred thoroughly, and then heated to 105-200° C., and the boiling time lasts for 2-6 hours. As the heating temperature increases, the pressure inside the sealed container gradually increases to form a high temperature and high pressure environment in the sealed container.

5. The method for extracting GLP-4 from Ganoderma lucidum polysaccharide according to claim 4, characterized in that: In step S2, the mixed liquid of Ganoderma lucidum powder and water in the sealed container is heated to 105-170° C., and the boiling time is continued for 3-6 hours. As the heating temperature increases, the pressure inside the sealed container gradually increases to form a high temperature and high pressure environment of the sealed container.

6. The method for extracting GLP-4 from Ganoderma lucidum polysaccharide according to claim 5, characterized in that: In step S6, the concentrated lyophilized powder containing the active ingredient is added with pure water to be prepared into 40-80 mg / ml, and is separated by multiple column chromatography to obtain the concentrate.

7. The method for extracting GLP-4 from Ganoderma lucidum polysaccharide according to claim 6, characterized in that: In the step S4, the medicinal residues are mixed and soaked with a NaOH solution at a ratio of 1:10-1:40 at a temperature of 40-100° C. for 1-5 hours, wherein the concentration of NaOH is 0.05-0.5 mol / L.

8. The method for extracting GLP-4 from Ganoderma lucidum polysaccharide according to claim 7, characterized in that: In the step S1, the ganoderma lucidum is washed with clean water to remove the dust on the surface, and then dried at 105° C. The dried ganoderma lucidum is crushed, and the crushed ganoderma lucidum powder is larger than 60 mesh.

9. The method for extracting GLP-4 from Ganoderma lucidum polysaccharide according to claim 8, characterized in that: In the step S2, the mixed liquid in the sealed container is heated to 105°C, 110°C, 115°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, 170°C, 175°C, 180°C, 185°C, 190°C, 195°C or 200°C, and the boiling time is 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h. As the heating temperature increases, the pressure inside the sealed container gradually increases to form a high temperature and high pressure environment of the sealed container.

10. The use of Ganoderma lucidum polysaccharide GLP-4 according to claim 1 or 2, characterized in that: The Ganoderma lucidum polysaccharide GLP-4 has good water solubility and is easily absorbed by the human body. It has the effects of treating emphysema, pulmonary fibrosis and anti-tumor. In particular, the therapeutic effect on emphysema and pulmonary fibrosis is extremely excellent, which is significantly better than that of bifenidone. When used in combination with chemotherapy drugs, it can eliminate the toxic side effects of chemotherapy drugs on the human body and has a certain effect on controlling and reducing tumor masses and reducing and eliminating tumor cells.

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