Polysaccharide compound having clear molecular structure and being capable of eliminating toxic side effects of chemotherapeutic drugs

Abstract

The present invention is applicable to the technical field of plant extraction and separation. Provided is a polysaccharide compound having a clear molecular structure and an anti-tumor effect. The polysaccharide compound is used for the treatment of patients with advanced cancers (patients who have lost surgical conditions, have a survival time of only three to six months, and are still eligible for chemotherapy), and has a molecular formula of: (C30H50O25)n. The active ingredient of Ganoderma is extracted under high temperature and high pressure to obtain the pharmacophore polysaccharide, i.e., the polysaccharide compound. The polysaccharide having the pharmacophore has good water solubility, is easily absorbed by a human body, has an anti-tumor efficacy, and is capable of preventing human tumorigenesis. Particularly, when being used in combination with a chemotherapeutic drug, the polysaccharide can eliminate toxic side effects on a human body caused by the chemotherapeutic drug, control and reduce the tumor mass, and reduce and eliminate tumor cells, which effects are all supported by corresponding experimental data.

Description

This invention relates to the field of plant extraction and separation technology, particularly to the extraction of a polysaccharide compound from Ganoderma lucidum with a defined molecular structure that possesses antitumor efficacy and can mitigate the toxic side effects of chemotherapy drugs. Background Ganoderma lucidum is a type of fungal plant with a long history of medicinal use in China and Japan. Ganoderma lucidum has a plurality of complex active ingredients, and over 150 compounds have been isolated from it, such as polysaccharides, triterpenes, sterols, alkaloids, furan derivatives, amino peptides, and inorganic elements. Geographic location (longitude and latitude), seed variation, growth environment, and differences in temperature, humidity, and light intensity can significantly affect the content, proportion, and presence of an active ingredient referred to in this invention in Ganoderma lucidum. Summary of Invention This invention provides a polysaccharide compound with a defined molecular structure that has antitumor efficacy and can eliminate the toxic side effects of chemotherapy dings. This invention aims to address the unmet international challenges of treating patients with advanced cancer, where “patients with advanced cancer” are defined as: those who are no longer surgical candidates, have a life expectancy of only three to six months, and are still eligible for chemotherapy; the “challenges of treating patients” refer to: allowing patients with advanced cancer to regain their appetite quickly (typically within two to three weeks), reducing or stabilizing tumor size, and using in combination with chemotherapy drugs to essentially eliminate the toxic side effects induced by the chemotherapy drugs on the human body. Long-term combination use with the chemotherapy drugs can achieve the objective of substantially eradicating cancer cells or keeping the number of cancer cells within safe limits for high-quality human survival. Another important function of the active ingredients specified in this invention is the prevention of mutations in normal human cells and the prevention of cancer cell formation. Furthermore, the active ingredient in this invention, when used in combination with the chemotherapy drugs, exhibit exceptionally good therapeutic effects on lung cancer, liver cancer, and breast cancer, demonstrating a certain degree of broad-spectrum activity. The invention provides a method for extracting Ganoderma lucidum polysaccharide GLP-2, comprising the steps of: SI. dusting and drying Ganoderma lucidum, then crushing Ganoderma lucidum to make Ganoderma lucidum powder; S2. placing the crushed Ganoderma lucidum powder in a sealed container mixed with water, heating under high temperature and pressure to fully dissolve the Ganoderma lucidum powder with the water into a medicinal juice solution; S3, using membrane concentration technology to separate the medicinal juice solution to obtain a concentrated solution with an active ingredient and not fully dissolved medicinal residue; and S4. mixing the concentrated solution containing the active ingredient with pure water to a water solution with a preset concentration, followed by multiple column chromatography separations to obtain the Ganoderma lucidum polysaccharide GLP-2 with the active ingredient. A further aspect of the invention: In step S2, the mixture of the Ganoderma lucidum powder and the water in the sealed container is fully stirred and heated at a high temperature to 105-200°C, with boiling time lasting for 2-6 h, during which the internal pressure in the sealed container gradually increases as the heating temperature rises, forming a high-temperature and high-pressure environment within the sealed container. A further aspect of the invention: In step S2, the mixture of the Ganoderma lucidum powder and the water in the sealed container is heated at a high temperature to 105-170°C, with boiling time lasting for 3-6 h, during which the internal pressure in the sealed container gradually increases as the heating temperature rises, forming the high-temperature and high-pressure environment within the sealed container. A further aspect of the invention: In step S4, the concentrated liquid containing the active ingredient is mixed with the pure water at a concentration ratio of 1:2 to 1:5. A further aspect of the invention: In step S3, Ganoderma lucidum residue is removed from the extracted water solution containing the active ingredient by using the membrane concentration technology, obtaining a concentrated liquid or paste containing the active ingredient. A further aspect of the invention: In step SI, the Ganoderma lucidum is rinsed with clean water to remove surface dust and dried at 105°C, and the dried Ganoderma lucidum is crushed, with the crushed Ganoderma lucidum powder being larger than 60 mesh. A further aspect of the invention: In step S2, the mixed liquid in the sealed container is heated at a high temperature 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, with boiling times of 2 h, 2.5 h, 3 h, 3.5 h, 4 h. 4.5 h. 5 h, 5.5 h, or 6 h, during which the internal pressure in the sealed container gradually increases as the heating temperature rises, forming the high-temperature and high-pressure environment within the sealed container. The invention also provides the Ganoderma lucidum polysaccharide GLP-2, wherein the structural formula of the Ganoderma lucidum polysaccharide GLP-2 is where n = 92-147. A further aspect of the invention: n is 92, 96, 100, 105, 110, 115, 120, 125, 130, 135, 140, 144, or 147. This invention also provides the application of the Ganoderma lucidum polysaccharide GLP-2. The Ganoderma lucidum polysaccharide GLP-2 is characterized by its water solubility, is readily absorbed by the human body, offers antitumor effects, and has proven strong efficacy in the prevention of tumor development in humans. Notably, when used in combination with chemotherapy drugs, it can alleviate toxic side effects caused by the chemotherapy drugs on the human body, control and reduce tumor masses, and reduce and eliminate cancer cells. The beneficial effects of this invention include a simple extraction process, high polysaccharide yield, low production cost, and ease of operation. The resultant Ganoderma lucidum polysaccharide is highly soluble and readily absorbed by the human body, enhancing its antitumor effects. Brief Description of the Drawings Figure 1: Flowchart of the method for extracting Ganoderma lucidum polysaccharide GLP-2, provided by an embodiment of the invention. Figure 2: Schematic diagram of IgMp-RT (peak molecular weight) calibration curves provided by an embodiment of the invention. Figure 3: Schematic diagram of IgMp-RT (weight-average molecular weight) calibration curvew provided by an embodiment of the invention. Figure 4: Schematic diagram of IgMp-RT (number-average molecular weight) calibration curves provided by an embodiment of the invention. Figure 5: Schematic diagram of the molecular weight of Ganoderma lucidum polysaccharide GLP-2, provided by an embodiment of the invention. Figure 6: Ion chromatography 1 of mixed standard 16 sugars, provided by an embodiment of the invention. Figure 7: Ion chromatography 2 of mixed standard 16 sugars, provided by an embodiment of the invention. Figure 8: GCMS chromatogram of the Ganoderma lucidum polysaccharide GLP-2 (PMAA) provided by an embodiment of the invention. Figure 9: Schematic diagram 1 of the mass-to-charge ratio analysis results of permethylated alditol acetate (PMAA) of polysaccharide, provided by an embodiment of the invention. Figure 10: Schematic diagram 2 of the mass-to-charge ratio analysis results of permethylated alditol acetate (PMAA) of polysaccharide, provided by an embodiment of the invention. Figure 11: Schematic diagram 3 of the mass-to-charge ratio analysis results of permethylated alditol acetate (PMAA) of polysaccharide, provided by an embodiment of the invention. Figure 12: Schematic diagram 4 of the mass-to-charge ratio analysis results of permethylated alditol acetate (PMAA) of polysaccharide, provided by an embodiment of the invention. Figure 13: Schematic diagram 5 of the mass-to-charge ratio analysis results of permethylated alditol acetate (PMAA) of polysaccharide, provided by an embodiment of the invention. Figure 14: Schematic diagram of the hydrogen spectrum provided by an embodiment of the invention. Figure 15: Schematic diagram of the carbon spectrum provided by an embodiment of the invention. Figure 16: Schematic diagram of the Deptl35 spectrum provided by an embodiment of the invention. Figure 17: Schematic diagram of HH-COSY provided by an embodiment of the invention. Figure 18: Schematic diagram of HSQC provided by an embodiment of the invention. Figure 19: HMBC spectrum provided by an embodiment of the invention. Figure 20: Schematic diagram of NOESY provided by an embodiment of the invention. Figure 21: Schematic diagram of the control group of LLC-bearing mouse model treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 22: Schematic diagram of the cisplatin group of LLC-bearing mice treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 23: Schematic diagram of the cisplatin + low-dose GLP-2 group of LLC-bearing mice treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 24: Schematic diagram of the cisplatin + high-dose GLP-2 group of LLC-bearing mice treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 25: Schematic diagram of tumor models in the control group of LLC-bearing mice treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 26: Schematic diagram of tumors in the cisplatin group of LLC-bearing mice treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 27: Schematic diagram of tumors in the cisplatin + low-dose GLP-2 group of LLC-bearing mice treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 28: Schematic diagram of tumors in the cisplatin + high-dose GLP-2 group of LLC-bearing mice treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 29: Schematic diagram of the control group of H22-bearing mouse model treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 30: Schematic diagram of the cisplatin group of H22-bearing mice treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 31: Schematic diagram of the cisplatin + low-dose GLP-2 group of H22-bearing mice treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 32: Schematic diagram of the cisplatin + high-dose GLP-2 group of H22-bearing mice treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 33: Schematic diagram of the control group of H22-bearing mouse tumor model treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 34: Schematic diagram of the cisplatin group of H22-bearing mouse tumor treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 35: Schematic diagram of the cisplatin + low-dose GLP-2 group of H22-bearing mouse tumor treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 36: Schematic diagram of the cisplatin + high-dose GLP-2 group of H22-bearing mouse tumor treated with Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy, provided by an embodiment of the invention. Figure 37: Schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on the tumor of orthotopic H22-bearing mice (binning: 8 x 8; T = 30 s), provided by an embodiment of the invention. Figure 38: Schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on the tumor of orthotopic H22-bearing mice, provided by an embodiment of the invention. Figure 39: Schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on the liver of orthotopic H22-bearing mice (x200), provided by an embodiment of the invention. Figure 40: Schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on the spleen of orthotopic H22-bearing mice (x200), provided by an embodiment of the invention. Figure 41: Schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on the stomach of orthotopic H22-bearing mice (x200), provided by an embodiment of the invention. Figure 42: Schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on the kidneys of orthotopic H22-bearing mice (x200), provided by an embodiment of the invention. Detailed Description of the Embodiments Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments below described by reference to the drawings are exemplary and are intended for the purpose of explaining the invention and should not be construed as limiting the scope of the invention. As shown in Figure 1, the present invention provides a flowchart for the method for extracting the Ganoderma lucidum polysaccharide GLP-2, described in detail as follows: In step SI, harvested Ganoderma lucidum, either raw or having undergone preliminary processing, is washed with clean water in washing equipment to remove surface dust. After dust removal, the Ganoderma lucidum is transferred to drying equipment for high-temperature drying at a temperature of 105°C. The dried Ganoderma lucidum is then placed in a crusher or mill to be broken down into Ganoderma lucidum powder. The crushed Ganoderma lucidum powder is sieved, and particles of the Ganoderma lucidum powder larger than 60 mesh are screened out, while particles of the Ganoderma lucidum powder smaller than 60 mesh are returned to the crusher or mill for further crushing. This process is repeated multiple times until the crushed Ganoderma lucidum powder meets specified requirements. In step S2, the Ganoderma lucidum powder that meets the requirements is mixed with pure water and placed in a sealed container. The sealed container is heated, in which the temperature is continuously increased to create a high-temperature, high-pressure environment, facilitating the thorough dissolution of the Ganoderma lucidum powder with the water to form a mixed solution. The sealed container is heated to a temperature between 105°C and 200°C, with a boiling time of 2-6 h, preferably between 105°C and 170°C, for 3-6 h. More preferably, the heating occurs at temperatures of l()5°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, with a boiling time of 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, or 6 h, allowing the thorough dissolution. The sealed container is a reaction kettle. In step S3, the water solution extracted, containing the active ingredient, is centrifuged to remove residues. The medicinal liquid is then concentrated using membrane concentration technology to obtain a concentrated liquid. In step S4, the concentrated liquid containing the active ingredient is prepared at a specific concentration, separated by column chromatography, and then concentrated and lyophilized to obtain Ganoderma lucidum polysaccharide GLP-2 with the active ingredient. This method offers a simple extraction process, high polysaccharide yield, low production costs, and simple operation. The invention also provides the Ganoderma lucidum polysaccharide GLP-2, wherein the structural formula of the Ganoderma lucidum polysaccharide GLP-3 is the molecular formula is (CsoHsoChsK where n = 92-147. nis 92, 96, 100, 105, 110, 115, 120, 125, 130, 135, 140, 144, or 147. Following the acquisition of the Ganoderma lucidum polysaccharide GLP-2 with the aforementioned structure, assay experiments were conducted and the following results are reported herein. I. Molecular Weight Determination 1. Experimental Objective To determine the molecular weight and purity of the polysaccharide using HPGPC. 2. Experimental Materials 2.1 Equipment Equipment name Manufacturer Model High-performance liquid chromatography Shimadzu LC-10A Differential refractometer Shimadzu RI-10A BRT105-104-102 tandem gel permeation chromatography column BoRui Saccharide BRT 105-104-102 (8 * 300 mm ) Electronic scale Sartorius CPA225D Centrifuge Eppendorf Eppendorf5424 Pipette Sartorius 200 uL, 1,000 uL 2.2 Materials Reagent Manufacturer Batch No. Catalog No. Grade Shelf life NaCl ACROS A0356762 139725000 ACROS 2022 2.3 Standards Standard Manufacturer Batch No. Storage conditions Purity Shelflife Dextranstandards 1152 Yuanye Bio-Technology A16A8L41850 Seal and store 99% 2 years 5000 Sigma 102084138 Seal and store >99% 2 years 11600 Sigma 102136543 Seal and store 99% 2 years 23800 Sigma 102124529 Seal and store >97% 2 years 48600 Sigma 102104509 Seal and store >99% 2 years 80900 Sigma 102108375 Seal and store >98% 2 years 148000 Sigma 102089360 Seal and store >98% 2 years 273000 Sigma 102110878 Seal and store 98% 2 years 409800 Sigma 102124507 Seal and store >98% 2 years 667800 Sigma 102104510 Seal and store >98% 2 years Molecular weight is measured in Daltons (Da). 3. Experimental Procedure 3.1 Reagent Preparation Reagent name Preparation method Storage conditions Shelf life 0.05M NaCl solution Precisely prepared, filtered through a 0.45-pm membrane, degassed by sonication for 10 min RT 1 month 3.2 Preparation of Sample and Standard Solutions Samples and standards were precisely weighed to prepare a 5 mg / mL solution, which was centrifuged at 12,000 rpm for 10 min, and the supernatant was filtered through a 0.22-pm micropore filter. Then the sample was transferred to a 1.8-mL sample vial. 3.3 Chromatographic Method Column: BRT105-104-102 tandem gel permeation chromatography column (8 x 300 mm); Mobile phase: 0.05M NaCl solution; Flow rate: 0.6 mL / min; Column temperature: 40°C; Injection volume: 20 uL; Detector: Differential refractometer RI-10 A. 4. Experimental Results As shown in Figures 2-4, calibration curves for IgMp-RT (peak molecular weight), IgMw-RT (weight-average molecular weight), and IgMn-RT (number-average molecular weight) were obtained. The equation of the calibration curves for IgMp-RT is: y = -0.1802x + 11.661R2 = 0.9928; The equation of the calibration curves for IgMw-RT is: y = -0.1926x + 12.241R2 = 0.9965; The equation of the calibration curves for IgMn-RT is: y = -0.1783x + 11.506R2 = 0.9911; Using the standard curves, a formula was derived to calculate the molecular weight of each sample. The molecular weight chromatograms for the samples are shown in Figure 5, with the results detailed in the table below. Sample ID RT (min) IgMp IgMw IgMn Mp Mw Mn Peak Area Ratio (%) 37.199 5.0 5.1 4.9 90728 119254 74717 100 Note: The peak at 46.5 min corresponds to the mobile phase. II. Monosaccharide Composition Determination Experiment 1. Experimental Objective To determine the monosaccharide composition using an ion chromatograph. 2. Experimental Principle This method is based on the electrochemical activity of sugar molecules and their ionization in strong alkaline solutions. Sugar compounds are weak acids with a pKa greater than 11. In high pH eluents, they partially or fully exist as anions. Efficient anion exchange and separation of sugar compounds is achieved, which leverages differences in ion exchange due to variations in pKa of different sugars, as well as differences in hydrophobic interactions between some sugars and the anion exchange resin. Detection is then accomplished by measuring the current produced by the oxidation of hydroxyl groups in the sugar molecules at a gold electrode surface. 3. Experimental Materials 3.1 Equipment Equipment name Manufacturer Model Ion chromatograph ThermoFisher ICS5000 Electric thermostatic blast drying oven LICHEN 101-1BS Nitrogen blower LICHEN UGC-24M Electronic scale Sartorius BS210S Centrifuge ThermoFisher D-37520 Pipette DRAGONLAB 19050983 3.2 Reagents Reagent Manufacturer Batch No. Catalog No. Grade Trifluoroacetic acid ACROS A0356762 139725000 AR 50% sodium hydroxide solution Alfa Aesar Z21E036 33382 GR Sodium acetate ThermoFishe 191126 059326 GR 3.3 Standards Standard Manufacturer Batch No. Storage conditions Purity Mannose Bo Saccharide Rui C17D9H77586 Seal and store AR Rhamnose Bo Saccharide Rui H10S9Z69863 Seal and store AR Galacturonic acid Bo Saccharide Rui K02A9B66077 Seal and store AR Galactose Bo Saccharide Rui El927035 Seal and store AR Glucose Bo Saccharide Rui Q18F10N80946 Seal and store AR Glucuronic acid Bo Saccharide Rui K14M10S82777 Seal and store AR Arabinose Bo Saccharide Rui S15A10G85850 Seal and store AR Xylose Bo Saccharide Rui A22S6X3606 Seal and store AR Fucose Bo Saccharide Rui X29D7Y27768 Seal and store AR Glucosamine hydrochloride Bo Saccharide Rui A22S6X3606 Seal and store AR N-acety 1-D-gl ucosamine Bo Saccharide Rui A21J8X40372 Seal and store AR D-fructose Bo Saccharide Rui J01J10R89818 Seal and store AR D-ribose Bo Saccharide Rui H26F10Z81556 Seal and store AR Galactosamine hydrochloride Bo Saccharide Rui B01J8S37079 Seal and store AR L-guluronic acid Bo Saccharide Rui S200115AG1 Seal and store >98% D-mannuronic acid Bo Saccharide Rui S200108AM1 Seal and store >98% 4. Experimental Methods 4.1 Reagent Preparation Reagent name Preparation method Storage conditions 15 mM NaOH solution 2.4 g of 50% NaOH solution, 2 L of water RT 15 mM NaOH &100 mM NaOAc solution 1.2 g of 50% NaOH solution, 8.2 g of NaOAc, 1 L of water RT 4.2 Preparation and Calculation Method for Standard Solutions Standard stock solutions were prepared using 16 different monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, galactosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, and mannuronic acid). Concentration standards of each monosaccharide standard solution were accurately prepared and used as a mixed standard. The mass of different monosaccharides was determined using an absolute quantification method and the molar ratios were calculated based on the molar mass of each monosaccharide. 4.3 Sample Preparation 5 mg of the sample was accurately weighed in an ampule. 2 mL of 3M TFA was added and hydrolyzed at 120°C for 3 h. The acid hydrolysate was accurately transferred to a tube and evaporated to dryness under nitrogen. 5 mL water was added, vortexed to mix, and then 50 pL was taken and added to 950 pL of deionized water, and centrifuged at 12,000 rpm for 5 min. The supernatant was transferred for IC analysis. 4.4 Chromatographic Method Column: Dionex CarbopacTM PA20 (3 x 150 mm); Mobile phase: A: H2O; B: 15 mM NaOH; C: 15 mM NaOH &100 mM NaOAc; Flow rate: 0.3 mL / min; Injection volume: 5 pL; Column temperature: 30°C; Detector: Electrochemical detector. 4.5 Standard Series No. Name ppm Name RT Area 1 Fucose 5 Fuc 5.659 18.741 2 Galactosamine hydrochloride 3 GalN 10.084 23.888 3 Rhamnose 5 Rha 10.475 10.717 4 Arabinose 3.7 Ara 11.092 16.035 5 Glucosamine hydrochloride 5 GlcN 12.367 31.057 6 Galactose 5 Gal 13.767 17.597 7 Glucose 5 Glc 15.484 20.442 8 N-acetyl-D-glucosamine 5 5 GlcNAc 16.792 13.652 9 Xylose 5 Xyl 17.834 22.737 10 Mannose 5 Man 18.117 14.734 11 Fructose 15 Fru 20.534 12.857 12 Ribose 10 Rib 22.484 26.868 13 Galacturonic acid 5 GalA 45.125 8.815 14 Guluronic acid 10 GulA 45.950 20.824 15 Glucuronic acid 5 GlcA 48.509 11.689 16 Mannuronic acid 10 ManA 50.992 22.847 C (standard) / A (standard) = C (sample) / A (sample) 5. Experimental Results Mixed standard: Solvent peaks at 2.0 min for sodium hydroxide and at 41 min for sodium acetate, as shown in Figures 6 and 7. Name RT Molar Ratio Fucose 5.959 0.000 Galactosamine hydrochloride 10.817 0.000 Rhamnose 11.334 0.000 Arabinose 11.792 0.000 Glucosamine hydrochloride 13.384 0.000 Galactose 14.609 0.000 Glucose 16.759 0.789 N-acetyl-D-ghicosamine 18.55 0.000 Xylose 19.209 0.000 Mannose 19.825 0.000 Fructose 22.259 0.000 Ribose 24.309 0.000 Galacturonic acid 44.592 0.000 Guluronic acid 45.092 0.042 Glucuronic acid 47.284 0.168 Mannuronic acid 50.2 0.000 III. Experiment on the Determination of Polysaccharide Linkage 1. Experimental Objective To determine the linkage patterns of polysaccharide samples through derivatization such as methylation by GC-MS analysis. 2. Experimental Materials 2.1 Equipment Equipment name Manufacturer Model Rotary evaporator Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd. R-1001VN Nitrogen blower LICHEN UGC-24M Magnetic stirrer DLAB MS7-H550-Pro Vacuum drying oven LICHEN 101-1BS Gas chromatograph-mass spectrometer Agilent 6890-5973 2.2 Reagents Reagent Manufacturer Batch No. Catalog No. Grade Trifluoroacetic acid ACROS A0356762 139725000 AR Methyl iodide Adamas Pl345479 01111630 AR Sodium borohydride Aldrich MKCD7945 205591 AR Ethyl acetate Vokai 08050003 40065982 AR Acetic anhydride f f l TO T TT oUoiii 20170314 10000318 AR Perchloric acid Aldrich SHBF7833V 311421 AR Acetic acid Fisher 156174 A35-500 AR Methanol Merck 10941735810 67-56-1 AR Sodium hydroxide HUSHI 20150429 10019718 AR Dimethyl sulfoxide Adamas P1265087 759270 AR Sodium hydride Adamas P1306059 81778A AR Methylation kit Borui Saccharide BRT-2020JJH BRT-JJH AR 3. Experimental Methods 3.1 Reagent Preparation Reagent name Preparation method Storage conditions 3M trifluoroacetic acid IV trifluoroacetic acid + 3 V water Store in refrigerator at 5°C Sodium hydride dry powder 60% sodium hydride washed with hexane Store dry at room temperature Sodium borodeuteride and sodium hydroxide solution 20 mg + 20 mM NaOH solution Seal and store 20% acetic acid methanol solution IV glacial acetic acid + 4 V water Store in refrigerator at 5°C Polysaccharide methylation kit A: anhydrous alkaline solution Store in refrigerator at 5°C B: methyl iodide solution 3.2 Sample Methylation The sample underwent methylation, hydrolysis, and acetylation, followed by GC-MS analysis, which was compared with the standard mass spectral library. 2-3 mg of the polysaccharide sample was weighed and placed in a glass reaction vial, 1 mL of anhydrous DMSO was added, and methylation reagent A was added rapidly. The solution was sealed and dissolved under ultrasonication, then methylation reagent B was added. The reaction was allowed at 30°C in a magnetic stirring water bath for 60 min. Finally, 2 mL of ultrapure water was added to stop the methylation reaction. The methylated polysaccharide was taken, 1 mL of 2M trifluoroacetic acid (TFA) was added, and hydrolyzed for 90 min. The rotary evaporator was used to dry. 2 mL of double-distilled water was added to the residue, which was reduced with 60 mg of sodium borohydride for 8 h, neutralized with glacial acetic acid, and evaporated using the rotary evaporator. Then, it was dried in a 101 °C oven, then 1 mL of acetic anhydride was added for acetylation and reacted at 100°C for 1 h, and cooled. Then 3 mL of toluene was added, vacuum concentrated to dry, and this process was repeated 4-5 times to remove excess acetic anhydride. The acetylated product was dissolved in 3 mL of CH2CI2 and transferred to a separatory funnel. A small amount of distilled water was added and shaken thoroughly, then the upper aqueous layer was removed. This process was repeated four times. The CH2CI2 layer was dried with an adequate amount of anhydrous sodium sulfate, brought to a volume of 10 mL, and placed in a vial for liquid analysis. The acetylated sample was analyzed using a Shimadzu GCMS-QP 2010 gas chromatograph-mass spectrometer. GC-MS Conditions: RXL5 SIL MS column 30 m x 0.25 mm x 0.25 pm; Temperature program: started at 120°C, increased at 3°C / min to 250°C, held for 5 min; Injector temperature at 250°C, detector temperature at 250°C, carrier gas was helium, flow rate was 1 mL / min. 4. Experimental Results The GC-MS chromatogram of the sample (PMAA) is shown in Figure 8. The permethylated alditol acetate (PMAA) analysis of the polysaccharide is presented in the following table and Figures 9-13. RT Methylated sugar Mass fragments (m / z) Molar ratio Type of linkage 17.412 2,3. 4, 6-Me.t-Glcp 45,71, 87, 101, 117, 129, 145, 161,205 0. 261 Glcp-(1 — 21. 343 2, 4. 6-Me3-Glcp 45, 71, 87, 101, 117, 129, 161, 189, 233 0. 243 -►3) -Glcp- (!-► 21.935 2, 3, 6-.Me3-Glcp 45, 87, 99, 101, 113, 117, 129, 131, 161. 173, 233 0. 171 -»4) -Glcp- (l-» 22. 752 2, 3, 4-Me.-Glcp 45, 87, 99. 101, 117, 129, 161, 189, 233 0. 167 -»6-Glcp-(l — 27. 58 2, 3-Me2-Glcp 45,71,85,87,99, 101, 117, 127, 159.201 0. 158 —4, 6)-Glcp-n- IV. NMR Spectral Analysis and Interpretation 1. Experimental Materials and Equipment Deuterium oxide (D2O, 99.9%) and deuterated acetone as internal standard; freeze-dryer, Bruker 600M Nuclear Magnetic Resonance (NMR) spectrometer; 2. Experimental Procedure 50 mg of the polysaccharide sample was weighed and dissolved in 0.5 mL of deuterium oxide followed by freeze-drying. The lyophilized powder was redissolved in 0.5 mL of deuterium oxide and freeze-drying was continued. The process was repeated to ensure complete exchange of labile hydrogens. Subsequently, the sample was dissolve in 0.5 mL of deuterium oxide, and 1H NMR, 13C NMR, DEPT135 one-dimensional and two-dimensional spectral measurements were performed at 25°C using a 600 MHz NMR spectrometer. 3. Experimental Results The hydrogen spectrum signals were primarily concentrated between 3.0 and 5.5 ppm. The signals for sugar ring protons were between 83.2-4.0 ppm, with main terminal group protons peaks at 84.66, 4.64, 4.46, 4.43, and 4.41, primarily distributed in the 4.3-5.5 ppm region as shown in Figure 14. Carbon spectral analysis in 13C NMR (201 MHz, D2O): The NMR carbon spectrum signals were mainly concentrated between 60-120 ppm. Observations of the carbon spectrum indicated main anomeric carbon signal peaks at 8103.92, 103.88, 103.82, 103.81, and 103.11, primarily between 893-105. Other notable signal peaks were at 874.51, 76.73, 70.88, 76.4, 70.48, 74.75, 85.63, 69.62, 77.02, 62.03, 74.15, 71.56, 80.14, 76.67, 61.66, 76.68, 74.08, 81.22, 75.39, 70.19, 72.71, 74.54, 69.22, 76.07, and 62.12 ppm. Based on monosaccharide composition results, the polysaccharide is composed of glucose, indicating the polysaccharide is primarily glucan. This is depicted in Figure 15. Deptl35 spectral analysis showed inverted peaks at 70.48, 70.19, 62.12, 62.03, and 61.66 ppm, indicative of chemical shifts for C6, as shown in Figure 16. Figures 17-20 present HSQC spectra where the anomeric carbon signal at 8103.11 corresponds to anomeric hydrogen signal at 84.64. HH-COSY identifies Hl-2 signal at 4.64 / 3.51; H2-3 signal at 3.51 / 3.67; H3-4 signal at 3.67 / 3.73. We deduce Hl, H2, H3, and H4 as 84.64, 3.51, 3.67, and 3.73 respectively, corresponding to 8103.11,72.71, 74.54, and 69.22. Deptl35 analysis confirmed C6 at 62.12 ppm, with H6b at 3.83 ppm and H6a at 3.63 ppm, correlating to C5 at 76.07; thus, these signals are attributed to the glycosidic bond P-Glcp-(1—>. HSQC spectra show that the anomeric carbon signal at 8103.94 corresponds to anomeric hydrogen signal at 84.66. HH-COSY identifies Hl-2 signal at 4.66 / 3.47; H2-3 signal at 3.47 / 3.65. We deduce Hl, H2, and H3 as 84.66, 3.47, and 3.65 respectively, corresponding to 8103.88, 74.75, and 85.63. HH-COSY identifies H6b-a signal at 3.81 / 3.62; H6a-5 signal at 3.62 / 3.39. The corresponding H6b, H6a, and H5 are at 83.81,3.62, and 3.39 respectively. The corresponding C5 is at 77.02; the chemical shift of C6 is at 862.03. Therefore, this signal is attributed to the glycosidic bond —>3)-p-Glcp-(l—>. Further HSQC observations show anomeric carbon signal at 5103.92, corresponding to anomeric hydrogen signal at 54.41. HH-COSY identifies Hl-2 signal at 4.41 / 3.22; H2-3 signal at 3.22 / 3.38; H3-4 signal at 3.38 / 3.54. We deduce Hl, H2, H3, and H4 as 54.41, 3.22, 3.38, 3.54 respectively, corresponding to Cl-4 at 5103.92, 74.51, 76.73, and 70.88. NOESY spectra show correlated peaks at 54.43 with 3.38, 3.54, 3.76, and 4.12. Deptl35 combined with HSQC allows the attribution of 53.76,4.12 as peaks forH6a,b; H5 at 3.54 ppm. Corresponding C5 is at 576.40; C6 chemical shift is at 570.48, with 116a at 53.76,4.11. Therefore, this signal is attributed to the glycosidic bond -^6)-P-Glcp-( 1—►. Using similar patterns and combining HMBC and NOESY, all glycosidic bond signals are assigned as shown in the following table: Hydrogen and Carbon Signal Attribution Glycosyl residues Hl / Cl H2 / C2 H3 / C3 H4C4 H5 C5 H6a C6 H6b p-D-Glcp-( 1 —► 4.64 3.51 3.67 3.53 3.64 3.63 3.83 103.61 71.61 74.54 69.22 76.07 62.12 -3)-p-D-Glcp-(l^ 4.66 3.47 3.65 3.41 3.42 3.65 3.83 103.88 74.75 85.63 69.62 77.26 62.03 —p”D'”CjrlCjp“(^ 1 4.41 3.22 3.38 3.34 3.54 3.76 4.12 103.92 74.51 76.73 70.88 76.25 70.48 ^4.6)-p-D-Glcp-(W 4.46 3.30 3.66 3.61 3.82 4.13 3.77 103.81 74.08 76.68 81.22 76.62 70.19 ^4)-p-D-Glcp-(W 4.43 3.28 3.55 3.57 3.42 3.84 3.68 103.82 74.15 75.56 80.14 76.67 61.66 Main Chain Analysis: From the HMBC spectra, based on the one-dimensional and two-dimensional NMR spectra, we have attributed the signals of the glycosidic bonds in the polysaccharide: The anomeric hydrogen of the glycosidic bond —>6)-|3-D-Glcp-(l—> shows correlation signal peaks with C6 of ^4,6)-p-D-Glcp-(l^, indicating the presence of a —>6)-P-D-Glcp-(l—>4,6)-P-DGlcp-(l —> linkage. Branch Chain Analysis: From the HMBC spectra, the anomeric hydrogen of the glycosidic bond P-D-Glcp-(1 >shows correlation peaks with the H3 of —>3)-p-D-Glcp-( 1>, indicating the presence of a p-D-Glcp-( 1 ->3)-p-D-Glcp-( 1 linkage. The anomeric hydrogen of the glycosidic bond —>3)-P-D-Glcp-(l—> shows correlation peaks with the H4 of —>4)-P-D-Glcp-(l—►, indicating the presence ofa —>3)-P-D-Glcp-(l—>4)-P-D-Glcp-(l—»linkage. The anomeric hydrogen of the glycosidic bond —>4)-p-D-Glcp-( 1^ shows correlation peaks with the H4 of ^4,6)-p-D-Glcp-(l^, indicating the presence of a ^4)-p-D-Glcp-(1^4,6)-p-D-Glcp-(l^ linkage. Based on the above, we can deduce that the main chain of the polysaccharide is the p-1,6 glucan, with p-D-Glcp-(l—>3)-p-D-Glcp-(l—>4)-p-D-Glcp-(l—» linked to the main chain through an O-4 bond of —>4,6)-0-D-Glcp-(l—>. The condensed structural formula is as follows. TO d <-> TO th ■8 d O J 4 -»6)-3-D-Glcp-{1^6)-P-D-Glcp-(l^ o This invention also provides the application of the Ganoderma lucidum polysaccharide GLP-2. The Ganoderma lucidum polysaccharide GLP-2 is characterized by its water solubility, is readily absorbed by the human body, offers antitumor effects, and has proven efficacy in the prevention of tumor development in humans. Notably, when used in combination with cisplatin, a chemotherapy drug, it can alleviate toxic side effects caused by the chemotherapy drugs on the human body, control and reduce tumor masses, and reduce and eliminate cancer cells. The Ganoderma lucidum polysaccharide GLP-2 is used in medications to inhibit tumor metastasis or to enhance human immune function. Experimental Study on the Antitumor Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on LLC-Bearing Mice and Study Data Experimental Objective To study the antitumor effect of Ganoderma lucidum polysaccharide GLP-2 on lung cancer-bearing mice which are prepared by implanting LLC tumor homogenate in the right axilla of C57 mice. This study aims to provide experimental evidence for clinical studies of GLP-2. Experimental Materials Test Sample Ganoderma lucidum polysaccharide GLP-2, provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd. Positive Control Cisplatin, batch number: E2128081, product of Shanghai Aladdin Biochemical Technology Co., Ltd. Laboratory Animals 68 SPF male C57 mice, weighing 16-18 g, supplied by Guangdong Medical Laboratory Animal Center. Laboratory animal production license number: SCXK (Yue) 2022-0002; laboratory animal quality certification number: 44007200109382. Main Reagents PBS buffer, prepared by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.; fetal bovine serum, product of Zhejiang Tianhang Biotechnology Co., Ltd.; DMEM culture medium, product of Gibco; 0.25% trypsin, product of Gibco. Main Instruments Vernier caliper, product of Shanghai Tool Works Co., Ltd.; 1-2000 scale, Dongguan Nancheng Changxie Electronic Products Factory; ophthalmic scissors and tweezers, products of Shanghai Jinzhong Medical Instrument Co., Ltd.; CCL-170B-8 CO2 incubator, product of ESCO, Singapore; Luna-II cell counter, product of Nanjing Hengqiao Instrument Co., Ltd. Experimental Methods Healthy LLC cells were inoculated subcutaneously into the left shoulder of 8 healthy male C57 mice. Once tumors reached a volume of 2,000-3,000 mm3, they were harvested and homogenized to prepare a homogenate suspension. This suspension was then injected subcutaneously into the axilla of 52 healthy male C57 mice to establish a solid tumor model. Once all mouse tumors averaged a volume of about 180 mm3, mice were randomized into groups based on tumor volume and were administered the respective drugs or drug solvents via oral gavage or intraperitoneal injection for 18 consecutive days. Longest and shortest diameters of tumors were measured every three days to calculate tumor volume, and mouse weights were recorded every three days. At the end of the experiment, tumors, spleens, and thymuses were harvested and weighed to calculate tumor, spleen, and thymus indices. Dosage Design Based on previous experimental results, Ganoderma lucidum polysaccharide GLP-2 was administered at a low dose of 50 mg / kg and a high dose of 150 mg / kg. The doses for each respective drug administered in this experiment are shown in Table 1. Rationale for cisplatin dosage design: Based on the clinical dosage of cisplatin, which should not exceed 100 mg / m2 per person per day, and considering the tolerance of mice to cisplatin, a dose of 4 mg / kg has been selected as the administration dosage. Table 1: Experimental groups and dosage design Group Dose (mg kg) Method of administration Administration volume (mL 10 g) Frequency of adnunistrauon Model control group Oral gavage 0.2 Once daily Cisplatin 4 Intraperitoneal injection. 0.2 Once everv 3 days Cisplatin + GLP-2 low-dose group 4+50 Intraperitoneal injection + oral gavage 0.2+0.2 Cisplatin, once every 3 days: GLP-2. once daily Cisplatin GLP-2 high-dose group 4+150 Intraperitoneal injection + oral gavage 0 2-0.2 Cisplatin, once every 3 days: GLP-2. once daily Nonnal group - Oral gavage 0.2 Once dailv Test Indicators Efficacy Indicators Relative tumor growth inhibition rate Relative tumor growth inhibition rate (%) = (1- TRTV / CRTV) x 100%. Where TRTV is the relative tumor volume in the experimental group, and CRTV is the relative tumor volume in the model control group. Relative tumor volume (RTV) = Vt / V0, where Vt is the tumor volume on day t of dosing, and V0 is the tumor volume at the time of grouping. Evaluation criteria: A relative tumor growth inhibition rate of >40% and a statistical analysis with P <0.05 indicate effective inhibition. Tumor growth inhibition rate Tumor growth inhibition rate (%) = (1-T / C)x 100%. Where T represents the average tumor weight in the treatment group, and C represents the average tumor weight in the model control group. Evaluation criteria: A tumor growth inhibition rate of >40% and a statistical analysis with P <0.05 indicate effective inhibition. Spleen and thymus organ coefficients: After the last dose, spleen, thymus, and tumor weights are measured, and organ coefficients are calculated. Tumor index (%) = (tumor weight I body weight) x 100%. Immune organ index (mg / g) = (organ mass / body weight) x 1000. Data Processing and Statistical Analysis Statistical analyses were performed using SPSS 17.0, with the significance level set at P <0.05. Measurement data were expressed as mean ± standard deviation (x-5). Normality and homogeneity of variance were tested using Leven’s test. If data met normality and homogeneity of variance (P >0.05), one-way ANOVA and LSD test were used for statistical analysis. If data did not meet normality and homogeneity of variance (P <0.05), the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P <0.05), comparison analysis was performed using Dunnett’s Test (a non-parametric method). Evaluation considered statistical differences and biological significance. Experimental Results Animal Mortality As shown in Table 2, the mortality rate was 25% in the cisplatin group and 0 in the remaining groups of mice. Table 2: Statistics on the number of surviving animals and mortality rate in each group Group Total number of animals Number of deaths Mortality rate (%) Survival time (days) Model control group 8 0 0 18=0 Cisplatin 8 2 25 17 1=19 Cisplatin + GLP-2 low-dose group 8 0 0 18=0 Cisplatin ~ GLP-2 high-dose group 8 0 0 18=0 Normal group 8 0 0 18=0 Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on Body Weight of LLC-Bearing Mice As shown in Table 3, compared to the normal group, the body weight of mice in the model control group significantly increased from DI 5 to DI8, and the body weight of mice in the cisplatin group, the cisplatin + GLP-2 low-dose and high-dose groups significantly decreased from D6 to D18. Compared to the model control group, the body weight of mice in the cisplatin group significantly decreased from D9 to DI 8, and the body weight of mice in the cisplatin + GLP-2 low-dose and high-dose groups significantly decreased from D6 to DI8. Compared to the cisplatin group, the body weight of mice in the cisplatin + GLP-2 low-dose group significantly decreased from D9 to D18, and the body weight of mice in the cisplatin + GLP-2 high-dose group significantly decreased on DI8. Table 3: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on body weight of LLC-bearing mice ( x ± s) Group Weight (g) DO D3 D6 D9 D12 D15 D18 Model control group 20.8*1.2 21.1*1.7 21.3*1.6 22.3*1.6 23.5*1.9 25.4*2.1* 26.2*2.2* Cisplatin 20.8*1.4 20.7*1.3 20.4*1* 19.7±0.8'+ 18.5±0.6*+ 18.2*1.2*+ 17.9*1.4*+ Cisplatin + GLP-2 low-dose group 20.9* 1 20.3*0.6 19.4±0.8‘+ 18.3*0.8** 16.6*0.8** 15.6*0.7** 14.7*1** Cisplatin + GLP-2 high-dose group 20.9*0.9 20.4*1.1 20*1.3** 19.3*1.8*+ 18.5*1.9*+ 17.3*1.7’* 16.3*1.4** Normal group 20.7*1.2 21.3*1 21.6*1.2 21.9*1.3 22.5*1.2 22.9±1.3+ 23*1.6+ Note: Compared to the model control group, +P <0.05; Compared to the cisplatin group, #P <0.05; Compared to the normal group, *P <0.05. Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on Tumor Volume in LLC-Bearing Mice As shown in Table 4, compared to the model control group, the tumor volume in the cisplatin group, the cisplatin + GLP-2 low-dose group, and the cisplatin + GLP-2 high-dose group was significantly reduced from D6 to DI 8. Compared to the cisplatin group, the tumor volume in the cisplatin + GLP-2 low-dose group and cisplatin + GLP-2 high-dose group was significantly reduced from DI 5 to DI 8. Table 4: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on tumor volume in LLC-bearing mice (x ± s) Tumor volume (mm3) uwup DO D3 D6 D9 D12 D15 DIS Model control group 183.1=35.1 411.7=104.7 1108.6=191.9 1462.8=233.1 2538.44485.7 3969.5=591 5493=528 Cisplatin 193=41.9 35O.8±138.5 692.44215.3* 956.44394* 1186.64386.4* 1388.94448.2* 1679.84439* Cisplatin GLP-2 low-dose 195 4=44 1 368.1=141.7 566.8*281.2- 735.24309.5* 799.34304’ 831.9i302.6-* 860.4=35’.—° group Cisplatin + GLP-2 high-dose 181 8=33 8 345.4=143.4 520.14271.9* 777.44480.6* 8894509* 847.9i391.1* 937.4±261* group Note: Compared to the model control group, +P <0.05; Compared to the cisplatin group, #P <0.05. Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on Relative Tumor Growth Inhibition Rate in LLC-Bearing Mice As indicated in Table 5, compared to the model control group, the relative tumor growth inhibition rate in the cisplatin group from D6 to D18 was over 40%, specifically 41.7%, 40.1%, 56.2%, 68.0%, and 70.9%; the relative tumor growth inhibition rate in the cisplatin + GLP-2 low-dose group from D6 to D18 was over 50%, specifically 52.7%, 54.0%, 71.1%, 80.8%, and 85.5%; the relative tumor growth inhibition rate in the cisplatin + GLP-2 high-dose group from D6 to DI 8 was over 45%, specifically 53.7%, 48.7%, 66.2%, 79.3%, and 83.3%. As shown in Table 6, compared to the cisplatin group, the relative tumor growth inhibition rate in the cisplatin + GLP-2 low-dose group from DI 5 to DI 8 was 39.9% and 50.3% (P <0.05), and the relative tumor growth inhibition rate in the cisplatin + GLP-2 high-dose group from DI 5 to DI 8 was 35.4% and 42.5% (P <0.05). Table 5: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on relative tumor growth inhibition rate in LLC-bearing mice (vs. the model control group) Relative tumor growth inhibition rate (compared to model Group control group) (%) D3 D6 D9 D12 D15 D18 Model control group - - - - - - Cisplatin 21.2 41.7+ 40.1+ 56.2+ 68.0+ 70.9+ Cisplatin + GLP-2 low-dose group 17.8 52.7+ 54.0+ 71.1* 80.8+ 85.5* Cisplatin + GLP-2 high-dose group 17.8 53.7+ 48.7+ 66.2+ 79.3+ 83.3+ Note: Compared to the model control group, +P <0.05. Table 6: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on relative tumor growth inhibition rate in LLC-bearing mice (vs. the cisplatin group) Group Relative tumor growth inhibition rate (compared to cisplatin group) (%) D3 D6 D9 D12 D15 D18 Cisplatin - - - - - - Cisplatin + GLP-2 low-dose group -4.4 18.9 23.1 34.0 39.9* 50.3* Cisplatin + GLP-2 high-dose group -4.4 20.6 14.3 22.8 35.4* 42.5* Note: Compared to the cisplatin group, *P <0.05. Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on Organ Coefficients and Tumor Growth Inhibition Rates in LLC-Bearing Mice As indicated in Table 7, compared to the model group, the tumor index, spleen index, and thymus index were significantly reduced in the cisplatin group, the cisplatin + GLP-2 low-dose group, and the cisplatin + GLP-2 high-dose group. Compared to the cisplatin group, the tumor index and spleen index were significantly reduced in the cisplatin + GLP-2 low-dose group and the cisplatin + GLP-2 high-dose group. Compared to the normal group, the spleen index was significantly higher in the model control group, but significantly reduced in the cisplatin + GLP-2 low-dose group; the thymus index was significantly reduced in the model control group, cisplatin group, cisplatin + GLP-2 low-dose group, and cisplatin + GLP-2 high-dose group. Compared to the model control group, the tumor growth inhibition rate was 67.3% in the cisplatin group, and 83.6% and 81.2% in the cisplatin + GLP-2 low-dose and high-dose groups, respectively. Compared to the cisplatin group, the tumor growth inhibition rate was 49.7% and 42.4% in the cisplatin + GLP-2 low-dose and high-dose groups, respectively. Table 7: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on organ indices and tumor growth inhibition rates in LLC-bearing mice Group Tumor index (%) Spleen index (mg / g) Thymus index (mg / g) Tumor growth inhibition rate (%) vs. the model control group Tumor growth inhibition rate (%) vs. the cisplatin group Model control group 24.7±4 1L4±2.2* l.l±0.4* - - Cisplatin 11.8±3.6+ 4.1±1.2+ o.4±o.r+ 67.3 - Cisplatin + GLP-2 low-dose group 7±3.6+# 2.1±0.3‘- o.4±o.r+ 83.6 49.7 Cisplatin + GLP-2 high-dose group 7.3±2.8+* 2.6±0.5+* o.4±o.r+ 81.2 42.4 Nonnal group - 3.1±0.3+ L7±0.2' - - Note: Compared to the model control group, +P <0.05; Compared to the cisplatin group, *P <0.05; Compared to the normal group, *P <0.05. Figures 21-24 show images of the tumor-bearing mice, corresponding to the groups as follows: Figure 21: model control group, Figure 22: cisplatin group, Figure 23: cisplatin + GLP-2 low-dose group, Figure 24: cisplatin + GLP-2 high-dose group. Figures 25-28 show images of the tumors in the tumor-bearing mice, corresponding to the groups as follows: Figure 25: model control group. Figure 26: cisplatin group, Figure 27: cisplatin + GLP-2 low-dose group, Figure 28: cisplatin + GLP-2 high-dose group. Conclusion Ganodernia Lucidum Polysaccharide GLP-2 combined with cisplatin significantly inhibits tumor growth in LLC-bearing mice and exhibits a significant synergistic effect. Experimental Study on the Antitumor Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on H22-Bearing Mice and Study Data Experimental Objective To study the antitumor effect of Ganoderma lucidum polysaccharide GLP-2 on hepatoma-bearing mice which are prepared by implanting H22 hepatoma tumor homogenate in the right axilla of C57 mice. This study aims to provide experimental evidence for clinical studies of GLP-2. Experimental Materials Test Sample Ganoderma lucidum polysaccharide GLP-2. provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd. Positive Control Cisplatin, batch number: E21268081, product of Shanghai Aladdin Biochemical Technology Co., Ltd. Laboratory Animals 55 SPF male C57 mice, weighing 16-18 g, supplied by Guangdong Medical Laboratory Animal Center. Laboratory animal production license number: SCXK (Yue) 2022-0002; laboratory animal quality certification number: 44007200104725. Main Reagents PBS buffer, prepared by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.; fetal bovine serum, product of Zhejiang Tianhang Biotechnology Co., Ltd.; 1640 culture medium, product of Gibco; 0.25% trypsin, product of Gibco. Main Instruments Vernier caliper, product of Shanghai Tool Works Co., Ltd.; 1-2000 scale, Dongguan Nancheng Changxie Electronic Products Factory; ophthalmic scissors and tweezers, products of Shanghai Jinzhong Medical Instrument Co., Ltd.; CCL-170B-8 COa incubator, product of ESCO, Singapore; Luna-II cell counter, product of Nanjing Hengqiao Instrument Co., Ltd. Experimental Methods A homogenate suspension of H22 mouse hepatoma was injected subcutaneously under the axilla of 45 healthy male C57 mice to create a solid tumor model. Once all mouse tumors averaged a volume of about 150 mm3, mice were randomized into groups based on tumor volume and were administered the respective drugs or drug solvents via oral gavage or intraperitoneal injection for 18 consecutive days. Longest and shortest diameters of tumors were measured every three days to calculate tumor volume, and mouse weights were recorded every three days. At the end of the experiment, tumors, spleens, and thymuses were harvested and weighed to calculate tumor, spleen, and thymus indices. Dosage Design Based on previous experimental results, Ganoderma lucidum polysaccharide GLP-2 was administered at a low dose of 50 mg / kg and a high dose of 150 mg / kg as shown in Table 8. Rationale for cisplatin dosage design: Based on the clinical dosage of cisplatin, which should not exceed 100 mg / m2 per person per day, and considering the tolerance of mice to cisplatin, a dose of 3 mg / kg has been selected as the administration dosage. Table 8: Experimental groups and dosage design Group Dose (mg "kg) Method of administration Administration volume (mD10 g) Frequency of administration Model control group - Oral gavage 0.2 Once daily Cisplatin 3 Intrapentoneal injection 0.2 Once every 3 days Cisplatin + GLP-2 low-dose group 3+50 Intraperitoneal injection + oral gavage 0 2^0 2 Cisplatin, once every 3 days: GLP-2. once daily Cisplatin + GLP-2 high-dose group 3+150 Intraperitoneal injection + oral gavage 0.2+0 2 Cisplatin, once every 3 days GLP-2. once daily Normal group - Oral gavage 0 2 Once daily Test Indicators Efficacy Indicators Relative tumor growth inhibition rate Relative tumor growth inhibition rate (%) = (1- Trtv / Crtv) x 100%. Where Trtv is the relative tumor volume in the experimental group, and Crtv is the relative tumor volume in the model control group. Relative tumor volume (RTV) = Vt / Vo, where Vt is the tumor volume on day t of dosing, and Vo is the tumor volume at the time of grouping. Evaluation criteria: A relative tumor growth inhibition rate of >40% and a statistical analysis with P <0.05 indicate effective inhibition. Tumor growth inhibition rate Tumor growth inhibition rate (%) = (1 - T / C) x 100%. Where T represents the average tumor weight in the treatment group, and C represents the average tumor weight in the model control group. Evaluation criteria: A tumor growth inhibition rate of >40% and a statistical analysis with P <0.05 indicate effective inhibition. Spleen and thymus organ coefficients: After the last dose, spleen, thymus, and tumor weights are measured, and organ coefficients are calculated. Tumor index (%) = (tumor weight / body weight) x 100%. Immune organ index (mg / g) = (organ mass / body weight) * 1000. Data Processing and Statistical Analysis Statistical analyses were performed using SPSS 17.0, with the significance level set at P <0.05. Measurement data were expressed as mean ± standard deviation (A ^5). Normality and homogeneity of variance were tested using Leven’s test. If data met normality and homogeneity of variance (P >0.05), one-way ANOVA and LSD test were used for statistical analysis. If data did not meet normality and homogeneity of variance (P <0.05), the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P <0.05), comparison analysis was performed using Dunnett’s Test (a non-parametric method). Evaluation considered statistical differences and biological significance. Experimental Results Animal Mortality As shown in Table 9, the mortality rate for all groups of mice was 0. Table 9: Statistics on the number of surviving animals and mortality rate in each group Group Total number of animals Number of deaths Mortality rate (%) Survival time (days) Model control group 8 0 0.0 18±0 Cisplatin 8 0 0.0 18±0 Cisplatin + GLP-2 low-dose group 8 0 0.0 1810 Cisplatin + GLP-2 high-dose group 8 0 0.0 1810 Normal group 8 0 0 1810 Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on Body Weight of H22-Bearing Mice As indicated in Table 10, compared to the normal group, the body weight of mice in the model control group significantly increased on D18; the body weight of mice in the cisplatin group significantly increased from D12 to DI 8; the body weight of mice in the cisplatin + GLP-2 low-dose group significantly decreased from D6 to DI8; and the body weight of mice in the cisplatin + GLP-2 high-dose group significantly decreased from D12 to D18. Compared to the model control group, the body weight of mice in the cisplatin group significantly decreased from D12 to DI 8, the body weight of mice in the cisplatin + GLP-2 low-dose group significantly decreased from D6 to DI8, and the body weight of mice in the cisplatin + GLP-2 high-dose group significantly decreased from D9 to D18. Compared to the cisplatin group, the body weight of mice in the cisplatin + GLP-2 low-dose group significantly decreased on D6, D9, D12, and D18. Table 10: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on body weight of H22-bearing mice ( x ± s) Group Weight (g) DO D3 D6 D9 D12 D15 D18 Model control group 19.710.8 20.411.5 21.111.6 22.111.8 23.312.1 23.312.4 25.912.6* Cisplatin 20.6H.3 20.811.3 2111.3 2111.5 20.7H .4+* 20.5H .4+* 19.9H.7+* Cisplatin + GLP-2 low-dose group 19.511.9 19.711.4 1911.6 18.7H.7”"* 18.6H.6+#* 18.7H.5+* 17.711.2'”' Cisplatin + GLP-2 high-dose group 20.7H.1 20.611.3 20.211.6 20.2H.5+‘ 20.3H.9+‘ 20H.9+* 19.6H.8+* Normal group 20.2±0.6 2110.9 21.111 21.711 22.411 22.911.1 23.411.1 Note: Compared to the model control group, P <0.05; Compared to the cisplatin group, "P <0.05; Compared to the normal group, *P <0.05. Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on Tumor Volume in H22-Bearing Mice As shown in Table 11, compared to the model control group, the tumor volume in the cisplatin group was significantly reduced from D12 to D18, and the tumor volume in the cisplatin + GLP-2 low-dose group and the cisplatin + GLP-2 high-dose group was significantly reduced from D6 to D18. Compared to the cisplatin group, the tumor volume in the cisplatin + GLP-2 low-dose group was significantly reduced from D15 to D18, and the tumor volume in the cisplatin + GLP-2 high-dose group was significantly reduced from D12 to D18. Table 11: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on tumor volume in H22-bearing mice ( x ± s) Group Tumor volume (mm3) DO D3 D6 D9 D12 D15 D18 Model control group 158.2=39.9 257.5=76.4 540.5=180.1 826 1±332 1 1438.2=779.3 1971 4±99£ 2 3091.1=1537.4 Cisplatin Cisplatin GLP-2 low-dose 167 9=42 2 205.9±122.5 417.4=228.3 577.3±420.2 798±676.5+ 952.7±711.r 1097±S053* group Cisplatin - GLP-"’ high-dose 1 36=39.6 297 3x1723 3«3.3±108.4- 458.94226.3* 539.6±2S8.6* 557.5±212- 659.8±407*« group 174.1=47.5 237.1±133.5 331.7H76.F 373.81191.4* 383.7±185.9*» 490.7±243.7- 551.5±225.4*« Note: Compared to the model control group, P <0.05; Compared to the cisplatin group, *P <0.05. Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on Relative Tumor Growth Inhibition Rate in H22-Bearing Mice As indicated in Tables 12 and 136, compared to the model control group, the relative tumor growth inhibition rate in the cisplatin group from DI2 to DI 8 was over 45%, specifically 47.29%, 52.99%, and 64.27%; the relative tumor growth inhibition rate in the cisplatin + GLP-2 low’-dose group from D9 to DI 8 was over 45%, specifically 47.69%, 64.57%, 72.10%, and 79.82%; the relative tumor growth inhibition rate in the cisplatin + GLP-2 high-dose group from D6 to DI8 was over 45%, specifically 45.85%, 60.34%, 76.86%, 77.87%, and 83.93%. Compared to the cisplatin group, the relative tumor growth inhibition rate in the cisplatin + GLP-2 low-dose group from DI5 to DI8 was 40.64% and 43.52% (P <0.05), and the relative tumor growth inhibition rate in the cisplatin + GLP-2 high-dose group from D12 to D18 was 56.10%, 52.93%, and 55.03% (P<0.05). Table 12: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on relative tumor growth inhibition rate in H22-bearing mice (vs. the model control group) Relative tumor growth inhibition rate (compared to model Group control group) (%) D3 D6 D9 D12 D15 D18 Model control group - - - - - - Cisplatin 30.90 25.54 33.52 47.29+ 52.99+ 64.27+ Cisplatin + GLP-2 low-dose group -5.78 3L59+ 47.69+ 64.57+ 72.10+ 79.82+ Cisplatin + GLP-2 high-dose group 22.01 45.85+ 60.34+ 76.86+ 77.87+ 83.93+ Note: Compared to the model control group, P ‘ <0.05. Table 13: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on relative tumor growth inhibition rate in H22-bearing mice (vs. the cisplatin group) Relative tumor growth inhibition rate (compared to Group cisplatin group) (%) D3 D6 D9 D12 D15 D18 Cisplatin - - - - - - Cisplatin + GLP-2 low-dose group -53.08 8.11 21.31 32.78 40.64* 43.52* Cisplatin + GLP-2 high-dose group -12.86 27.27 40.33 56.10* 52.93* 55.03* Note: Compared to the cisplatin group, *P <0.05. Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on Organ Coefficients and Tumor Growth Inhibition Rates in H22-Bearing Mice As indicated in Table 14, compared to the model group, the tumor index, spleen index, and thymus index were significantly reduced in the cisplatin group, the cisplatin + GLP-2 low-dose group, and the cisplatin + GLP-2 high-dose group. Compared to the cisplatin group, the tumor index was significantly reduced in the cisplatin + GLP-2 low-dose group and the cisplatin + GLP-2 high-dose group. Compared to the normal group, the spleen index was significantly reduced in the model control group; the thymus index was significantly reduced in the cisplatin group, the cisplatin + GLP-2 low-dose group, and the cisplatin + GLP-2 high-dose group. Compared to the model control group, the tumor growth inhibition rate was 57.3% in the cisplatin group, and 76.4% and 79.3% in the cisplatin + GLP-2 low-dose and high-dose groups, respectively. Compared to the cisplatin group, the tumor growth inhibition rate was 44.8% and 51.6% in the cisplatin + GLP-2 low-dose and high-dose groups, respectively. Table 14: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on organ indices and tumor growth inhibition rates in H22-bearing mice Group Tumor index (%) Spleen index (mg / g) Thymus index (mg / g) Tumor growth inhibition rate (%) vs. the model control group Tumor growth inhibition rate (%) vs. the cisplatin group Model control group 10.2±5.1 4.9±1.5* L3±0.3 - - Cisplatin 5.7±4.6+ 3.1±1+ 0.6±0.4+* 57.3+ - Cisplatin + GLP-2 low-dose group 3.6±1.6“ 2.8±L1+ 0.5±0.2+* 76.4+ 44.8# Cisplatin + GLP-2 high-dose group 2.9±1.6+# 2.6±0.1+ 0.8±0.2+* 79.3+ 51.6" Normal group - 2.3±0.1 1.4±0.2 - - Note: Compared to the model control group, +P <0.05; Compared to the cisplatin group, #P <0.05; Compared to the normal group, *P <0.05. Figures 29-32 show images of the tumor-bearing mice, corresponding to the groups as follows: Figure 29: model control group, Figure 30: cisplatin group, Figure 31: cisplatin + GLP-2 low-dose group, Figure 32: cisplatin + GLP-2 high-dose group. Figures 33-36 show images of the tumors in the tumor-bearing mice, corresponding to the groups as follows: Figure 33: model control group, Figure 34: cisplatin group, Figure 35: cisplatin + GLP-2 low-dose group, Figure 36: cisplatin + GLP-2 high-dose group. Conclusion Ganoderma Lucidum Polysaccharide GLP-2 combined with cisplatin significantly inhibits tumor growth in H22-bearing mice and exhibits a significant synergistic effect. Experimental Study on the Antitumor Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on Orthotopic H22-Bearing Mice and Study Data Experimental Objective To study the effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on orthotopic H22-bearing mice which are prepared by implanting 1122 hepatoma tumors in the right axilla of C57 mice. This study aims to provide experimental evidence for clinical studies of GLP-2. Experimental Materials Test Sample Ganoderma lucidum polysaccharide GLP-2, batch number: ALM20200518A1, provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd. Control Kanglaite soft capsules, batch number: 20211006, Zhejiang Kanglaite Pharmaceutical Co., Ltd.; cisplatin injection, batch number: 601211204, Jiangsu Hansoh Pharmaceutical Co., Ltd. Laboratory Animals 60 SPF-grade male C57 mice, weighing 12-15 g, provided by Hunan SJA Laboratory Animal Co., Ltd. Laboratory animal production license number: SCXK (Xiang) 2019-0004. The animals were housed in Barrier Environment Laboratory D of Hunan Puruima Pharmaceutical Research Center Co.. Ltd., under the laboratory animal use license number: SYXK (Xiang) 2020-0015. Main Reagents 0.9% sodium chloride injection, batch number: 21071401C, 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 manufactured by Wako Pure Chemical Industries, Ltd. of Japan. Main Instruments AR223CN Electronic Scale, Ohaus Instruments (Changzhou) Co., Ltd.; LABOSPECT003 Automatic Biochemical Analyzer, Hitachi, Japan; AniViewlOO Multimodal Animal In Vivo Imaging System, ANDOR; TDZ5-WS Benchtop Multi-Tube Automatic Balance Centrifuge, Hunan Kaida Industrial Development Co., Ltd.; ME2002E Electronic Scale, Shimadzu Corporation, Japan; Flow Cytometer, BD Biosciences; ASP200S Fully Automatic Tissue Dehydrator, ASP300S Fully Automatic Tissue Dehydrator, TP1020 Fully Automatic Dehydrator, Hl 1210 Slide Spreader, Hl 1220 Slide Dryer, RM2235 Paraffin Microtome, EG1150H+C Tissue Embedding Station, AutoStainer XL Automatic Slide Stainer + CV5030 Automatic Cover Slipper, BX43 Biological Microscope + MD50 Digital Imaging System, CX31 Biological Microscope, all from Leica, Germany. Experimental Methods Liver cancer (H22) cells, labeled with Luc fluorescent marker at a concentration of lx 107 cells / mL, were initially inoculated into the peritoneal cavity of 5 male C57 mice. After the development of ascites, the ascitic fluid was aseptically extracted, washed with HBSS buffer, centrifuged to discard the supernatant, and stained with Trypan Blue, and cells in the ascitic fluid were counted under a microscope. The cell concentration was adjusted to 1 x 1013 / mL with HBSS buffer and inoculated into the right hepatic region of 45 male C57 mice, at a volume of 10 pL per mouse, to prepare the orthotopic tumor-bearing mice. One week later, the liver tumor formation in mice was detected using a small animal in vivo imaging system. Based on tumor size, the mice were randomized into groups: model control group, cisplatin group (4 mg / kg), Kanglaite soft capsule group (1,404 mg / kg), cisplatin + Kanglaite soft capsule group (4 + 1,404 mg / kg), cisplatin + GLP-2 low-dose group (4 + 130 mg / kg), and cisplatin + GLP-2 high-dose group (4 + 1,170 mg / kg), with 6 mice per group. An additional 6 mice served as the normal control group. The normal control group and the model control group were administered pure water by oral gavage. The chemotherapy group received intraperitoneal injections of cisplatin, and the other groups were administered their respective drug solutions by oral gavage (20 mL / kg) or intraperitoneal injection (10 mL / kg), once daily for 14 consecutive days. After the last dose, blood was collected from the orbital plexus to test blood WBC, RBC, liver and kidney function indicators (ALT, AST, BUN, CRE), and CD3+ / CD4+, CD3+ / CD8+ ratios. The spleen, thymus, and tumors were weighed to calculate organ coefficients. Dosage Design Based on previous experimental results, Ganoderma lucidum polysaccharide GLP-2 was administered at a low dose of 130 mg / kg and a high dose of 1170 mg / kg as shown in Table 15. The proposed clinical dose for Kanglaite soft capsules is 0.45 g per capsule, 6 capsules per dose, 4 doses per day, which totals 10.8 g per day. When converted to an equivalent mouse dose based on body surface area, it is calculated as 10.8 g / day x 0.0026 / 0.02 kg = 1,404 mg / kg. This study used the proposed clinical dose as a basis for the experiment. Rationale for cisplatin dosage design: Based on the clinical dosage of cisplatin, which should not exceed 100 mg / m2 per person per day, and considering the tolerance of mice to cisplatin, a dose of 4 mg / kg has been selected as the administration dosage. Table 15: Experimental groups and dosage design Group Dose (mgkg) Method of administration Frequency of administration Normal control group - Oral gavage Once daily Model control group - Oral gavage Once daily Cisplatin group 4 Intraperitoneal injection Once every 3 days Kanglaite soft capsule group 1404 Oral gavage Once daily Cisplatin + Kanglaite soft capsule group 4+1404 Intraperitoneal injection e- oral gavage Cisplatin, once every 3 days: Kanglaite soft capsule, once daily Cisplatin + GLP-2 low-dose group 4+130 Intraperitoneal injection oral gavage Cisplatin, once ever}’ 3 days GLP-2, once daily Cisplatin - GLP-2 high-dose group 4+1170 Intraperitoneal injection ~ oral gavage Cisplatin, once every 3 days. GLP-2. once daily Test Indicators Efficacy Indicators Animal survival and general condition: Weight was recorded weekly, along with any deaths among the animals. Tumor volume measurement: Tumor volume changes were measured weekly using small animal in vivo imaging technology. Hematological tests: After the last dose, routine blood tests (WBC and RBC) and biochemical tests (liver and kidney functions) were performed. Immune organs: The thymus, spleen, tumor, and liver were weighed, and organ coefficients were calculated as follows: organ coefficient (%) = organ weight / fasting body weight x 100% CD4+ and CD8+ content measurement: After the last dose, lymphocyte subtypes CD3 / CD4' and CD3+ / CD8+ in the blood were measured using flow cytometry. Data Processing and Statistical Analysis Significant figures of the study data were rounded according to the nearest whole number. Statistical analysis was conducted in accordance with the center’s SOP, using SPSS software. Measurement data are expressed as mean ± standard deviation ( x ± s), and normality and homogeneity of variance were tested using Leven’s test. If not statistically significant (P >0.05), one-way ANOVA was used for statistical analysis. If ANOVA was statistically significant (P <0.05), the LSD test (parametric method) was used for comparison analysis. If the variance was not homogeneous (P <0.05), the Kruskal-Wallis test was employed. If the Kruskal-Wallis test was statistically significant (P <0.05), the Dunnett’s Test (non-parametric method) was used for comparison analysis. Statistical significance was determined at a = 0.05, where P <0.05 indicates statistical significance and P< 0.01 indicates highly significant differences. Experimental Results Animal Mortality As shown in Table 16, before administration, the mice exhibited normal activity, movement, and gait; after administration, a reduction in activity was observed, and tumor growth impacted their food and water intake, leading to minimal weight gain. In the model control group, 2M01 died on D12; in the cisplatin group, 3M01 / 3M05 and 3M02 died on D9 and D10, respectively; in the Kanglaite soft capsule group, 4M01 died on D9; in the cisplatin + Kanglaite soft capsule group, 5M01 and 5M06 died on D8 and DI 1, respectively; in the cisplatin + GLP-2 high-dose group, 7M03, 7M04, and 7M05 died on D8, D12, and DI 1. The mortality rates for each group were 0%, 16.7%, 50.0%, 16.7%, 16.7%, 0%, and 50.0%, respectively. The cisplatin group and the cisplatin + GLP-2 high-dose group had the highest mortality rate, both reaching 50.0%. Table 16: Statistics on the number of surviving animals and mortality rate in each group Group Dose (mg / kg) Number of animals Number of deaths Number of sunning animals Mortality rate (%) Normal control group - 6 0 6 0 Model control group - 6 1 5 16.7 Cisplatin group 4 6 3 3 50 0 Kanglaite soft capsule group 1404 6 1 5 16 7 Cisplatin + Kanglaite soft capsule group 4+1404 6 1 5 16.7 Cisplatin + GLP-2 low-dose group 4+130 6 0 6 0 Cisplatin + GLP-2 lugh-dose group 4+1170 6 3 3 50 0 Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on Body Weight of Orthotopic H22-Bearing Mice As shown in Table 17, compared to the normal control group, the body weight of mice in the model control group significantly decreased on W2 after administration (P< 0.01). Compared to the model control group, the body weight of mice in both the cisplatin group, the cisplatin + Kanglaite soft capsule group, the cisplatin + GLP-2 low-dose group, and the cisplatin + GLP-2 high-dose group significantly decreased on W1 and W2 after administration (P <0.05 or P <0.01). Compared to the cisplatin group, the body weight of mice in the Kanglaite soft capsule group significantly increased on W2 after administration (P <0.05 or P <0.01). Compared to the Kanglaite soft capsule group, the body weight of mice in the cisplatin + GLP-2 low-dose and high-dose groups significantly decreased on W1 and W2 after administration (P <0.05 or P <0.01). There was no significant statistical difference between treatment groups compared to the cisplatin ± Kanglaite soft capsule group. Table 17: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on body weight of orthotopic H22-bearing mice (X + 5) Group Weight (g) WO W1 W2 Normal control group 19.9 ± 1.2 18.9 ± 1.6 22.4 ± 1.7 Model control group 18.3 ±1.9 19.1 ± 1.9 19.6 ±1.8 Cisplatin group 18.5 ± 1.3 16.9 ±1.8* 15.1 ±1.6** Kanglaite soft capsule group 18.2 ± 1.5 18.7 ±2.9 19.0±1.3## Cisplatin + Kanglaite soft capsule group 18.6 ±0.9 15.5±2.3**&& 16.6±3.7**& Cisplatin + GLP-2 low-dose group 18.0± 1.0 16.7 ± 1.2*& 15.8 ± 1.5**&& Cisplatin + GLP-2 high-dose group 18.3 ± 1.1 15.3 ± 1.5**&& 14.1 ± 1.5**&& Note: Compared to the normal control group, P <0.01; compared to the model control group, *P <0.05, **P <0.01; compared to the cisplatin group, *P <0.05, ®#P <0.01; compared to the Kanglaite soft capsule group, &P <0.05, &&P <0.01; compared to the cisplatin + Kanglaite soft capsule group, *P <0.05, **P < o.oi. Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on Tumors in Orthotopic H22-Bearing Mice As shown in Figures 37, 38, and Table 18, compared to the normal control group, the tumors in the mice of the model control group significantly increased (P <0.01); compared to the model control group, the tumors in the mice of the cisplatin + GLP-2 low-dose group and the Kanglaite soft capsule group significantly decreased on W1 and W2 after administration (P <0.05 or P <0.01); the tumors in the mice of the cisplatin group and the cisplatin + Kanglaite soft capsule group significantly decreased on W2 after administration (P <0.05 or P <0.01). There was no statistical difference between the groups when compared to the cisplatin group. Compared to the Kanglaite soft capsule group, the tumors in the mice of the cisplatin + Kanglaite soft capsule group significantly increased on W1 after administration (P <0.05 or P< 0.01); the tumors in the mice of the cisplatin + GLP-2 high-dose group significantly increased on W1 and W2 after administration (P< 0.05). Compared to the cisplatin + Kanglaite soft capsule group, the tumors in the mice of the cisplatin + GLP-2 low-dose group significantly decreased on WI and W2 after administration {P <0.05); there was no statistically significant difference in the rest of the groups. As shown in Figure 37, the corresponding groups are as follows: A: normal group, B: model control group, C: cisplatin group, D: Kanglaite soft capsule group, E: cisplatin + Kanglaite soft capsule group, F: cisplatin + GLP-2 low-dose group, G: cisplatin + GLP-2 high-dose group. As shown in Figure 38, the corresponding groups are as follows: 1: normal group, 2: model control group, 3: cisplatin group, 4: Kanglaite soft capsule group, 5: cisplatin + Kanglaite soft capsule group, 6: cisplatin + GLP-2 low-dose group, 7: cisplatin + GLP-2 high-dose group. Table 18: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on tumors in orthotopic H22-bearing mice ( ¥ ± 5) Group WO W1 W2 Tumor (P^s'cm-'sr) Number of animals Tumor (P's-'cm-'sr) Number of animals Tumor (P-van- Ar) Number of animak Normal control group 0±0 6 0 = 0 6 0 = 0 6 Model control group 3897500±2651893** 6 17515000 ±2702626** 6 20012000 ±3407219“ 5 Cisplatin group 3756000±2439052 6 12898500 = 3588676 6 11871333 4 2^80016^ 3 Kanglaite soft capsule group 3888167 ±2341708 6 5906333±2'10545** 6 6040000 ±2159132** 5 Cisplatin -t- Kanglaite soft capsule 3968117 = 2511453 6 17516667 ±3438257 6 10266000 ± 2143720** group Cisplatin GLP-2 low-dose group 3751083=2310570 6 9081667 ± 2731045»* 6 4402333 ±1686743»»* 6 Cisplatin + GLP-2 high-dose group 3905167 ±2143597 6 13838333 ± 27780741 6 15633333 ± 21605041 3 Note: Compared to the normal control group, P <0.01; compared to the model control group, *P <0.05, **P <0.01; compared to the cisplatin group, "P <0.05, ##P <0.01; compared to the Kanglaite soft capsule group, &P <0.05, &&P <0.01; compared to the cisplatin + Kanglaite soft capsule group, *P< 0.05, **P <0.01. Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on Organ Coefficients and Tumor Growth Inhibition Rates in Orthotopic H22-Bearing Mice As shown in Table 19, compared to the normal control group, the organ coefficients of the spleen and liver tissues in the model control group significantly increased (P< 0.05 or P <0.01). Compared to the model control group, the thymus and spleen coefficients in both the cisplatin + GLP-2 low-dose and high-dose groups significantly decreased (P <0.05 or P <0.01), and the thymus and spleen coefficients in both the cisplatin group and the cisplatin + Kanglaite soft capsule group significantly decreased (P <0.05). There was no statistically difference across the treatment groups when compared to the cisplatin group. Compared to the Kanglaite soft capsule group, the thymus and spleen coefficients in the cisplatin + GLP-2 low-dose and high-dose groups significantly decreased (P<0.05 or P< 0.01), and the spleen coefficients in the cisplatin ± Kanglaite soft capsule group significantly decreased (P <0.05 or P <0.01); there was no statistically significant difference in the remaining treatment groups. Table 19: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on organ coefficients in orthotopic H22-bearing mice () Group Thymus coefficient Spleen coefficient Liver coefficient Normal control group 0.191 ±0.039 0.372 ±0.066 5.325 ±0.205 Model control group 0.191 ±0.162 1.151 ±0.713++ 12.497 ± 6.659+ Cisplatin group Kanglaite soft capsule 0.078 ±0.023* 0.524 ± 0.295* 11.365 ±3.599 group Cisplatin + Kanglaite soft 0.144 ±0.079 1.116 ±0.537 14.206 ±8.001 capsule group Cisplatin + GLP-2 low-dose 0.074 ± 0.063* 0.548 ± 0.351 *& 9.405 ±2.800 group 0.054 ± 0.036**& 0.440 ±0.119**&& 10.025 ±7.69 Cisplatin + GLP-2 high-dose group 0.044 ±0.038**& 0.412 ±0.314**&& 11.199 ±4.044 Note: Compared to the normal control group, P< 0.05, ++P <0.01; compared to the model control group, *P <0.05, **P <0.01; compared to the cisplatin group, *P <0.05; compared to the Kanglaite soft capsule group, &P <0.05, &&P< 0.01. Effect of Ganoderma Lucidum Polysaccharide GLP-2 Combined with Chemotherapy on Blood Biochemical Indicators in Orthotopic H22-Bearing Mice As shown in Table 20, compared to the normal control group, the blood AST and CRE level of mice in the model control group significantly increased (P <0.01); compared to the model control group, the blood WBC level of mice in the cisplatin + GLP-2 low-dose group and the Kanglaite soft capsule group significantly decreased (P <0.05), and blood BUN level of mice in the cisplatin + GLP-2 high-dose group significantly decreased (P <0.05). Compared to the cisplatin group, the blood BUN level in the mice of the cisplatin + GLP-2 high-dose group significantly decreased (P <0.05). Compared to the Kanglaite soft capsule group, the RBC level of mice in the cisplatin + GLP-2 low-dose and high-dose groups significantly decreased (P <0.05 or P <0.01). Compared to the cisplatin + Kanglaite soft capsule group, the blood WBC level in the mice of the cisplatin + GLP-2 high-dose group significantly increased (P <0.05 or P <0.01), with no statistical significance in other groups. Table 20: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on blood biochemical indicators in orthotopic H22-bearing mice (X ± S) Group WBC (10’,L) RBC (IO12!) ALT (U'L) AST (U'L) BUN (mmoL-L) CRE (umol / L) Normal control group 6 81 ±0 83 9 62 = 0 16 49 = 4 134= 18 8.10 ± 1 S3 10.5 = 0.9 Model control group 9 33 ± 1 29 9 18 = 0.44 84 = 31 761 ± 104“ 9.75= 1 51 19.8 = 2S~ Cisplatin group 4 4.1 = 183 8 91 = 0 22 115 = 39 771 . 153 13.52 = 4.94 189 = 2.7 Kanglaite soft capsule group 6.16 ± 1 60 8.80 = 0.15 72= 10 831=148 8.42= 1 86 15 1 = 1.7 Cisplatin + Kanglaite soft capsule 3.10 ±051* 8.54 = 0 2 / 63 = 21 431= 103 4.97=2.08 22.2 = 0.9 group Cisplatin = GLP-2 low-dose group 4.84 ± 0.75* 8.31 ± 0.21s 61 = 11 735 = 278 11.44 = 2.55 16.5 = 2.8 Cisplatin + GLP-2 high-dose group 5.29 ± 0.57* 7.80±0.64& 68 = 9 640=22 3.64 ±1.21*” 20 4 = 0.6 Note: Compared to the normal control group, +P <0.05, P <0.01; compared to the model control group, *P <0.05, **P <0.01; compared to the cisplatin group, #P <0.05, ^P <0.01; compared to the Kanglaite soft capsule group, &P <0.05, &&P <0.01; compared to the cisplatin + Kanglaite soft capsule group, *P <0.05, **P<0.01. As shown in Table 21, compared to the normal control group, there was a trend of increased blood (4)3'CD4 and CD37CD8' ratios in the model control group of mice, though the differences were not statistically significant. Compared to the model control group, the blood CD3+ / CD8+ ratio in the mice of the cisplatin + GLP-2 low-dose group significantly increased (P <0.05); the blood CD3 / CD4 and CD3+ / CD8+ ratios in the mice of the cisplatin + GLP-2 high-dose group significantly increased (P <0.05). Compared to the cisplatin group, the blood CD3+ / CD8+ ratio in the mice of the cisplatin + GLP-2 low-dose group significantly increased (P <0.05); the blood CD3+ / CD4+ and CD3+ / CD8+ ratios in the mice of the cisplatin + GLP-2 high-dose group significantly increased (P <0.05). There was no significant statistical difference between groups compared to the Kanglaite soft capsule group. There was no significant statistical difference between groups compared to the cisplatin + Kanglaite soft capsule group. Table 21: Effect of Ganoderma lucidum polysaccharide GLP-2 combined with chemotherapy on blood CD3+ / CD4+ and CD3+ / CD8+ ratios in orthotopic H22-bearing mice (X + 5) Group CD3+ / CD4+ CD3+ / CD8+ Normal control group 2.97 + 0.12 3.62 + 0.38 Model control group 4.76 + 1.37 4.73 + 0.75 Cisplatin group 4.26 + 0.04 4.21+0.81 Kanglaite soft capsule group 4.20 + 0.70 6.58 + 2.91 Cisplatin + Kanglaite soft capsule group 3.88 + 0.88 9.58 + 3.62 Cisplatin + GLP-2 low-dose group 6.23 + 1.00 11.70 + 1.50*# Cisplatin + GLP-2 high-dose group 10.45 + 2.74*# 16.81 ±3.40*# Note: Compared to the model control group, *P<0.05; compared to the cisplatin group, #P< 0.05; compared to the Kanglaite soft capsule group, &P <0.05; compared to the cisplatin + Kanglaite soft capsule group, *P <0.05. As shown in Figures 39-42, the mice in the model control group exhibited extensive hepatocellular carcinoma cell infiltration and necrosis, increased number of sinusoidal cells, and inflammatory cell infiltration in the liver; evident extramedullary hematopoiesis and diffuse red pulp in the spleen, liver cancer cell infiltration in the stomach and lesions on the serosa, with widespread atrophy of the gastric mucosa. After administration of Ganoderma lucidum polysaccharide GLP-2 combined with cisplatin, the extent of liver cancer cell infiltration and necrosis decreased in all groups, with increased splenic extramedullary hematopoiesis, and no significant lesions observed in the stomach and kidneys. As shown in Figures 39-42, the corresponding groups are as follows: A: normal group, B: model control group, C: cisplatin group, D: Kanglaite soft capsule group, E: cisplatin + Kanglaite soft capsule group, F: cisplatin + GLP-2 low-dose group, G: cisplatin + GLP-2 high-dose group Conclusion Ganoderma lucidum polysaccharide GLP-2 combined with cisplatin significantly inhibits the growth of tumors in orthotopic H22-bearing mice and demonstrates a notable synergistic effect. Discussion and Conclusion Liver cancer is a highly lethal malignancy, where current treatments involving surgical resection, chemotherapy, and radiotherapy merely delay symptoms, making complete cure extremely difficult. Liver cancer is notably resistant to chemotherapy, especially in cases of advanced liver cancer, where there is no reliable evidence proving that systemic chemotherapy can improve overall survival of patients with advanced liver cancer. The results of this study showed that in the model control group of mice, tumor volume significantly increased, spleen and liver indices significantly increased, red blood cell counts in the blood significantly increased, white blood cell counts significantly decreased, and liver and kidney functions were significantly abnormal. These findings indicate a decline in lymphatic system function during tumor progression in the model control group of mice. After the last dose, Ganoderma lucidum polysaccharide GLP-2 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, and significantly increased the number of white blood cells, with some recovery in liver and kidney function indicators. CD4+T cells and CD8+T cells mediate tumor immune responses, where CD8 T cells are the main effector cells of tumor immunity. Results indicate that Ganoderma lucidum polysaccharide GLP-2 combined with cisplatin significantly increased CD3+ / CD4+ and CD3+ / CD8+ ratios in the mice’s blood, suggesting that Ganoderma lucidum polysaccharide GLP-2 combined with cisplatin can protect immune organs and enhance the body’s own immune function, thereby playing a role in reducing toxicity and enhancing antitumor effects. Histopathological results also showed that Ganoderma lucidum polysaccharide GLP-2 can enhance the body’s immunity. Additionally, when compared to Kanglaite soft capsule combined with cisplatin, Ganoderma lucidum polysaccharide GLP-2 combined with cisplatin significantly reduced tumor, spleen indices, and thymus indices, indicating that Ganoderma lucidum polysaccharide GLP-2 combined with cisplatin has a stronger antitumor effect than Kanglaite soft capsule combined with cisplatin. The foregoing description concerns merely exemplary embodiments of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the invention should be included within the scope of the invention’s protection.

Claims

1. A type of Ganoderma lucidum polysaccharide GLP-2, characterized in that the molecular structural formula of the Ganoderma lucidum polysaccharide GLP-2 is; the molecular formula is: (C3oH5o02s)n; the5 main chain of the Ganoderma lucidum polysaccharide GLP-2 is P-1,6 glucan, with P-D-Glcp-(1—>3)-P-D-Glcp-(l—>4)-P-D-Glcp-(l—> linked to the main chain through an 0-4 bond of -M,6)-P-D-Glcp-(1—►; the condensed structural formula is:TP 6-84 / WTO6 ■CDTO 6 6 r>T5 i X 4-»6)-P-D-Glcp-(1^6)-p-D-Glcp-(l^, where n = 92-147.

2. The Ganoderma lucidum polysaccharide GLP-2 according to claim 1, whereinio n is selected from 92, 96, 100, 105, 110, 115, 120, 125, 130, 135, 140, 144, or 147.

3. A method for extracting the Ganoderma lucidum polysaccharide GLP-2 as claimed in claim 1 or 2, comprising the steps of:S1. dusting and drying Ganoderma lucidum, then crushing Ganoderma lucidum to make Ganoderma lucidum powder;S2. placing the crushed Ganoderma lucidum powder in a sealed container mixed with water, heating under high temperature and pressure to fully dissolve the Ganoderma lucidum powder with the water into a medicinal juice solution;S3, using membrane concentration technology to separate the medicinal juice solution to obtain a concentrated solution with an active ingredient and not fully dissolved medicinal residue; andS4. mixing the concentrated solution containing the active ingredient with pure water to a water solution with a preset concentration, followed by multiple column chromatography separations to obtain the Ganoderma lucidum polysaccharide GLP-2 with the active ingredient.

4. The method for extracting Ganoderma lucidum polysaccharide GLP-2 according to claim 3, characterized in that in step S2, the mixture of the Ganoderma lucidum powder and the water in the sealed container is fully stirred and heated at a high temperature to 105-200°C, with boiling time lasting for 2-6 h, during which the internal pressure in the sealed container gradually increases as the heating temperature rises, forming a high-temperature and high-pressure environment within the sealed container.

5. The method for extracting Ganoderma lucidum polysaccharide GLP-2 according to claim 3, characterized in that in step S2, the mixture of the Ganoderma lucidum powder and the water in the sealed container is heated at a high temperature to 105-170°C, with boiling time lasting for 3-6 h, during which the internal pressure in the sealed container gradually increases as the heating temperature rises, forming the high-temperature and high-pressure environment within the sealed container.

6. The method for extracting the Ganoderma lucidum polysaccharide GLP-2 according to claim 4 or 5, characterized in that in step S4, the concentrated liquid containing the active ingredient is mixed with the pure water at a concentration ratio of 1:2 to 1:5.

7. The method for extracting the Ganoderma lucidum polysaccharide GLP-2 according to claim 6, characterized in that in step S3, Ganoderma lucidum residue is removed from the extracted water solution containing the active ingredient by using the membrane concentration technology, obtaining a concentrated liquid or paste containing the active ingredient.

8. The method for extracting the Ganoderma lucidum polysaccharide GLP-2 according to claim 7, characterized in that in step S1, the Ganoderma lucidum is rinsed with clean water to remove surface dust and dried at 105°C, and the dried Ganoderma lucidum is crushed, with the crashed Ganoderma lucidum powder being larger than 60 mesh.

9. The method for extracting the Ganoderma lucidum polysaccharide GLP-2 according to claim 5, characterized in that in step S2, the mixed liquid in the sealed container is heated at a high temperature to 105°C, 110°C, 115°C, 125°C, 130°C, 135°C, 140°C, 145°C, 155°C, 160°C, 165°C, or 170°C, with boiling times of 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, or 6 h, during which the internal pressure in the sealed container gradually increases as the heating temperature rises, forming the high-temperature and high-pressure environment within the sealed container.

10. The method for extracting the Ganoderma lucidum polysaccharide GLP-2 according to claim 4, characterized in that in step S2, the mixed liquid in the sealed container is heated at a high temperature 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, with boiling times of 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, or 6 h, during which the internal pressure in the sealed container graduallyincreases as the heating temperature rises, forming the high-temperature and high-pressure environment within the sealed container.

11. The application of the Ganoderma lucidum polysaccharide GLP-2 and cisplatin in the preparation of anti-lung cancer and liver cancer drug products 5 according to claim 1 or 2, characterized by the combined use of the Ganoderma lucidum polysaccharide GLP-2 and the chemotherapy drug cisplatin, which can alleviate the toxic side effects caused by chemotherapy drugs on the human body, control and reduce tumor masses, and reduce and eliminate cancer cells.io

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