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 that has a clear molecular structure and an anti-tumor efficacy and that can eliminate toxic side effects of chemotherapy. 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). The molecular formula of the polysaccharide compound is: (C66H110O55)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

A polysaccharide compound with a clear molecular structure that can eliminate the toxic side effects of chemotherapy drugs Technical Field

[0001] The present invention belongs to the field of plant extraction and separation technology improvement, and in particular relates to a method and use for extracting a polysaccharide compound with a clear molecular structure, anti-tumor efficacy and the ability to eliminate the toxic side effects of chemotherapy drugs, namely, Ganoderma lucidum polysaccharide GLP-3, from plants. Background Art

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

[0003] Summary of the Invention

[0004] The purpose of the present invention is to provide a polysaccharide compound with a clear molecular structure, anti-tumor efficacy and the ability to eliminate the toxic side effects of chemotherapy.

[0005] The present invention aims to solve the treatment problem of advanced cancer patients that has not yet been solved internationally. The so-called advanced cancer patients are cancer patients who have lost the conditions for surgery, have only three to six months to survive, and are still eligible for chemotherapy. The so-called treatment problem is to enable advanced cancer patients to restore their appetite as quickly as possible (generally two to three weeks), shrink tumor masses or stop growing, and use them in combination with chemotherapy drugs to basically eliminate the toxic side effects of chemotherapy drugs on the human body. Long-term combined use with chemotherapy drugs can achieve the goal of basically eliminating cancer cells or controlling the number of cancer cells to a safe range for high-quality survival of the human body.

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

[0007] Another important function of the medicinal ingredients referred to in the present invention is that when used in combination with chemotherapy drugs, they show exceptionally excellent therapeutic effects on lung cancer, liver cancer, and breast cancer, showing a certain broad spectrum.

[0008] The present invention is achieved by providing a method for extracting GLP-3 from Ganoderma lucidum polysaccharide, the method comprising the following steps:

[0009] S1, removing dust from the Ganoderma lucidum, drying it, and then crushing it to prepare Ganoderma lucidum powder;

[0010] S2, placing the crushed Ganoderma lucidum in a sealed container, mixing with water and heating, and fully blending the Ganoderma lucidum powder and water into a medicinal juice solution under high temperature and high pressure;

[0011] S3, using membrane concentration technology to separate the medicinal juice solution to obtain a concentrated solution with medicinal components and incompletely fused medicinal residues;

[0012] S4. The concentrated solution containing the active ingredient is mixed with pure water in a ratio of a preset concentration to obtain an aqueous solution, which is subjected to multiple column chromatography separations and concentrated freeze-dried to obtain Ganoderma lucidum polysaccharide GLP-3 containing the active ingredient.

[0013] A further technical solution of the present invention is: in step S2, the Ganoderma lucidum powder and water are mixed and fully heated to 105-200° C., and the boiling time lasts for 2-6 hours. The Ganoderma lucidum powder and water in the sealed container are fully fused into a mixed medicinal liquid under a high temperature and high pressure environment.

[0014] A further technical solution of the present invention is: in step S2, the Ganoderma lucidum powder and water are mixed and fully heated to 105-170° C., and the boiling time lasts for 3-6 hours. The Ganoderma lucidum powder and water in the sealed container are fully fused into a mixed medicinal liquid under a high temperature and high pressure environment.

[0015] A further technical solution of the present invention is: in step S4, the concentrated solution containing the medicinal ingredients and pure water are prepared to a concentration ratio of 1:2-1:5.

[0016] A further technical solution of the present invention is: in step S3, the aqueous solution containing the extracted medicinal ingredients is concentrated by using membrane concentration technology to remove the Ganoderma lucidum residue therein to obtain a medicinal ingredient concentrated solution or paste.

[0017] A further technical solution of the present invention is: in step S1, the Ganoderma lucidum is rinsed with clean water to remove dust on the surface, dried at a high temperature of 105° C. in a drying device, and the dried Ganoderma lucidum is placed in a crushing device for crushing to obtain Ganoderma lucidum powder, wherein the Ganoderma lucidum powder is larger than 60 mesh.

[0018] A further technical solution of the present invention is: in step S2, the mixed liquid in the sealed container is heated to 105°C, 110°C, 115°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C or 170°C, and the boiling time is 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h, and the Ganoderma lucidum powder and water in the sealed container are fully fused into a mixed medicinal liquid under a high temperature and high pressure environment.

[0019] Another object of the present invention is to provide a Ganoderma lucidum polysaccharide GLP-3, the structural formula of which is Molecular formula (C 66 H 110O 55 ) n , where n=30-46.

[0020] A further technical solution of the present invention is: n is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46.

[0021] Another object of the present invention is to provide an application of GLP-3, a polysaccharide of Ganoderma lucidum. The polysaccharide GLP-3 of Ganoderma lucidum has good water solubility, is easily absorbed by the human body, has anti-tumor efficacy and has a strong effect on preventing the occurrence of tumors in humans. Especially when used in combination with chemotherapy drugs, it can eliminate the toxic side effects of chemotherapy drugs on the human body and has the effect of controlling and reducing tumor masses and reducing and eliminating tumor cells.

[0022] The invention has the following beneficial effects: the method has simple extraction process, high polysaccharide extraction rate, low production cost and simple operation; the ganoderma lucidum polysaccharide has good solubility and is easily absorbed by the human body, thereby exerting its anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 is a flow chart of a method for extracting GLP-3, a polysaccharide of Ganoderma lucidum, provided in an embodiment of the present invention.

[0024] FIG2 is a schematic diagram of a 1gMp-RT (peak molecular weight) calibration curve provided in an embodiment of the present invention.

[0025] FIG3 is a schematic diagram of a 1gMp-RT (weight average molecular weight) calibration curve provided in an embodiment of the present invention.

[0026] FIG4 is a schematic diagram of a 1gMp-RT (number average molecular weight) calibration curve provided in an embodiment of the present invention.

[0027] FIG5 is a schematic diagram of the molecular weight spectrum of Ganoderma lucidum polysaccharide GLP-3 provided in an embodiment of the present invention.

[0028] FIG6 is a schematic diagram of an infrared spectrum of a polysaccharide provided in an embodiment of the present invention.

[0029] FIG7 is a schematic diagram of an ion chromatogram of a mixed standard of 16 sugars provided in an embodiment of the present invention.

[0030] FIG8 is a second schematic diagram of an ion chromatogram of a mixed standard of 16 sugars provided in an embodiment of the present invention.

[0031] FIG9 is a GCMS chromatogram of a sample (PMAA) provided in an embodiment of the present invention.

[0032] FIG10 is a schematic diagram 1 of the result analysis of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0033] FIG11 is a second schematic diagram of the analysis results of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0034] FIG12 is a third schematic diagram of the analysis results of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0035] FIG13 is a fourth schematic diagram of the analysis results of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0036] FIG14 is a fifth schematic diagram of the analysis results of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0037] FIG15 is a sixth schematic diagram of the analysis results of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0038] FIG16 is a schematic diagram of a hydrogen spectrum provided by an embodiment of the present invention.

[0039] FIG17 is a schematic diagram of a carbon spectrum provided by an embodiment of the present invention.

[0040] FIG18 is a schematic diagram of a Dept135 spectrum provided in an embodiment of the present invention.

[0041] FIG19 is a schematic diagram of HH-COSY provided by an embodiment of the present invention.

[0042] FIG20 is a HSQC diagram provided by an embodiment of the present invention.

[0043] FIG21 is an HMBC spectrum provided by an embodiment of the present invention.

[0044] FIG22 is a schematic diagram of NOESY provided by an embodiment of the present invention.

[0045] Figure 23 is a schematic diagram of the control group of LLC lung cancer tumor-bearing mouse model treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided in an embodiment of the present invention.

[0046] FIG24 is a schematic diagram of the cisplatin group of LLC lung cancer-bearing mice treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy according to an embodiment of the present invention.

[0047] Figure 25 is a schematic diagram of the cisplatin + GLP-3 low-dose group of LLC lung cancer-bearing mice treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided by an embodiment of the present invention.

[0048] Figure 26 is a schematic diagram of the cisplatin + GLP-3 high-dose group of LLC lung cancer-bearing mice treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided by an embodiment of the present invention.

[0049] Figure 27 is a schematic diagram of the control group of the LLC lung cancer tumor-bearing mouse model treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided in an embodiment of the present invention.

[0050] Figure 28 is a schematic diagram of the cisplatin group of LLC lung cancer-bearing mice treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy according to an embodiment of the present invention.

[0051] Figure 29 is a schematic diagram of the cisplatin + GLP-3 low-dose group for the treatment of LLC lung cancer tumor-bearing mice treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided by an embodiment of the present invention.

[0052] Figure 30 is a schematic diagram of the cisplatin + GLP-3 high-dose group for the treatment of LLC lung cancer tumor-bearing mice treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided by an embodiment of the present invention.

[0053] Figure 31 is a schematic diagram of the control group of the H22 liver cancer-bearing mouse model treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided in an embodiment of the present invention.

[0054] Figure 32 is a schematic diagram of the Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy in the cisplatin group of H22 liver cancer-bearing mice provided by an embodiment of the present invention.

[0055] Figure 33 is a schematic diagram of the cisplatin + GLP-3 low-dose group of H22 liver cancer-bearing mice treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided by an embodiment of the present invention.

[0056] Figure 34 is a schematic diagram of the Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy for the cisplatin + GLP-3 high-dose group of H22 liver cancer-bearing mice provided by an embodiment of the present invention.

[0057] Figure 35 is a schematic diagram of the appearance of the control group of the H22 liver cancer-bearing mouse tumor model treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided in an embodiment of the present invention.

[0058] Figure 36 is a schematic diagram of the appearance of the cisplatin group of H22 liver cancer-bearing mice treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided by an embodiment of the present invention.

[0059] Figure 37 is a schematic diagram of the appearance of the cisplatin + GLP-3 low-dose group in H22 liver cancer-bearing mice treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided by an embodiment of the present invention.

[0060] Figure 38 is a schematic diagram of the appearance of the high-dose cisplatin + GLP-3 group of H22 liver cancer-bearing mice treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided by an embodiment of the present invention.

[0061] Figure 39 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on H22 orthotopic liver cancer tumors in mice (binning: 8×8; T=30s) provided by an embodiment of the present invention.

[0062] Figure 40 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on H22 orthotopic liver cancer tumors in mice provided by an embodiment of the present invention.

[0063] Figure 41 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on the liver of mice with H22 in situ liver cancer provided by an embodiment of the present invention (×200).

[0064] Figure 42 is a schematic diagram (×200) showing the effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on the spleen of mice with H22 orthotopic liver cancer provided by an embodiment of the present invention.

[0065] Figure 43 is a schematic diagram (×200) showing the effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on the thymus of mice with H22 orthotopic liver cancer provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0066] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0067] As shown in FIG1 , the flow chart of the method for extracting GLP-3 from Ganoderma lucidum polysaccharide provided by the present invention is described in detail as follows:

[0068] Step S1, washing the harvested Ganoderma lucidum or Ganoderma lucidum that has undergone a preliminary simple treatment with clean water on a washing device to remove dust on the surface thereof, transferring the Ganoderma lucidum after the dust is removed to a drying device for high-temperature drying at a drying temperature of 105° C., placing the Ganoderma lucidum after high-temperature drying on a crusher or a pulverizer to crush it into Ganoderma lucidum powder, sieving the crushed Ganoderma lucidum powder particles larger than 60 mesh, and returning the Ganoderma lucidum powder particles smaller than 60 mesh to the crusher or pulverizer for re-crushing, and repeating this process multiple times until the crushed Ganoderma lucidum powder particles meet the specified requirements.

[0069] Step S2: mixing the qualified Ganoderma lucidum powder with pure water and placing the mixture in a sealed container, heating the sealed container. The temperature of the sealed container will continue to rise as the heating continues, so that a high temperature and high pressure environment is formed in the sealed container. In this environment, it is easier to fully fuse the Ganoderma lucidum powder and water to form a mixed liquid. The sealed container is heated to a temperature between 105° C. and 200° C. for a boiling time of 2-6 hours, preferably between 105° C. and 170° C. for a boiling time of 3-6 hours. It is more preferably heated to 105°C, 110°C, 115°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, 170°C, 175°C, 180°C, 185°C, 190°C, 195°C or 200°C, with a boiling time of 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h, so that the mixed liquid is fully fused, and the closed container is a reactor.

[0070] In step S3, the extracted aqueous solution containing the medicinal components is centrifuged to remove the residue, and the medicinal solution is concentrated using a membrane concentration technology to obtain a concentrated solution.

[0071] Step S4, the concentrated solution containing the medicinal component is prepared into a certain concentration, separated by column chromatography, concentrated and freeze-dried to obtain the medicinal component Ganoderma lucidum polysaccharide GLP-3.

[0072] The method has simple extraction process, high polysaccharide extraction rate, low production cost and simple operation.

[0073] Another object of the present invention is to provide a Ganoderma lucidum polysaccharide GLP-3, the structural formula of which is Molecular formula C 66 H 110 O 55 ) n , where n=30-46.

[0074] n is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46.

[0075] After obtaining the Ganoderma lucidum polysaccharide GLP-3 with the above structure, it was subjected to a determination experiment and the following reported results were obtained.

[0076] 2. Molecular weight determination

[0077] 1. Purpose of the experiment

[0078] The molecular weight and purity of the polysaccharide were determined by HPGPC.

[0079] 2. Experimental Materials

[0080] 2.1 Instruments

[0081] 2.2 Materials

[0082] 2.3 Standards

[0083] 3. Experimental Procedure

[0084] 3.1 Reagent preparation

[0085] 3.2 Preparation of sample and standard solutions

[0086] Accurately weigh the sample and standard, prepare the sample into a 5 mg / ml solution, centrifuge at 12000 rpm for 10 min, filter the supernatant with a 0.22 μm microporous filter membrane, and then transfer the sample to a 1.8 ml injection vial.

[0087] 3.3 Chromatographic method

[0088] Chromatographic column: BRT105-104-102 gel column in series (8×300 mm); mobile phase: 0.05 M NaCl solution; flow rate: 0.6 ml / min, column temperature: 40°C; injection volume: 20 μl; detector: differential detector RI-10A.

[0089] 4. Experimental Results

[0090] As shown in Figure 2-4, the calibration curves of 1gMp-RT (peak molecular weight), 1gMw-RT (weight average molecular weight), and 1gMn-RT (number average molecular weight) were obtained.

[0091] The equation of the 1gMp-RT calibration curve is: y = -0.184x + 11.752R 2 =0.9956;

[0092] The equation of the lgMw-RT calibration curve is: y = -0.1961x + 12.315R 2 =0.9934;

[0093] The equation of the lgMn-RT calibration curve is: y = -0.1818x + 11.589R 2 =0.992;

[0094] Based on the standard curve, a calculation formula was derived to calculate the molecular weight of each sample. The molecular weight spectrum of the samples is shown in Figure 5, and the calculation results are shown in the table below.

[0095] Among them, 46.2min is the peak of the mobile phase.

[0096] 2. Monosaccharide composition determination experiment

[0097] 1. Purpose of the experiment

[0098] The monosaccharide composition was determined using ion chromatography.

[0099] 2. Experimental Principle

[0100] Based on the electrochemical activity of carbohydrate molecules and their ionization in strong alkaline solutions, carbohydrate compounds are weak acids with a pKa greater than 11. In high-pH eluents, they exist partially or completely as anions. By taking advantage of the differences in ion exchange caused by the pKa of different carbohydrate compounds and the different hydrophobic interactions between certain carbohydrates and anion exchange resins, efficient anion exchange separation of carbohydrate compounds is achieved. The current generated by the oxidation reaction of hydroxyl groups in the carbohydrate molecular structure on the gold electrode surface is then detected.

[0101] 3. Experimental Materials

[0102] 3.1 Instruments

[0103] 3.2 Reagents

[0104] 3.3 Standards

[0105] 4. Experimental Methods

[0106] 4.1 Reagent preparation

[0107] 4.2 Preparation and calculation method of standard solution

[0108] Sixteen monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid, mannuronic acid) were prepared into standard stock solutions.

[0109] Prepare a standard concentration of each monosaccharide standard solution as a mixed standard. Determine the mass of each monosaccharide using the absolute quantification method, and calculate the molar ratio based on the molar mass of the monosaccharides.

[0110] 4.3 Sample preparation

[0111] Accurately weigh 5 mg of sample into an ampoule, add 2 ml of 3M TFA, and hydrolyze at 120°C for 3 h. Accurately transfer the acid hydrolysis solution to a tube and blow dry with nitrogen. Add 5 ml of water and vortex to mix thoroughly. Aspirate 50 μL and add 950 μL of deionized water. Centrifuge at 12,000 rpm for 5 min. Collect the supernatant for IC analysis.

[0112] 4.4 Chromatographic methods

[0113] Chromatographic column: Dionex Carbopac TM PA20 (3*150mm); mobile phase: A: H2O; B: 15mMNaOHC: 15mMNaOH & 100mM NaOAC; flow rate: 0.3ml / min; injection volume: 5μL; column temperature: 30℃; detector: electrochemical detector.

[0114] 4.5 Standard Sequence

[0115] C(standard) / A(standard)=C(sample) / A(sample)

[0116] 5. Experimental Results

[0117] Mixed standard: Solvent peak: 2.0 min is the peak of sodium hydroxide, 41 min is the peak of sodium acetate, as shown in Figures 6 and 7.

[0118] 3. Polysaccharide Linkage Determination Experiment

[0119] 1. Purpose of the experiment

[0120] The polysaccharide samples were derivatized by methylation and then their connection patterns were determined by GC-MS.

[0121] 2. Experimental Materials

[0122] 2.1 Instruments

[0123] 2.2 Reagents

[0124] 3. Experimental Methods

[0125] 3.1 Reagent preparation

[0126] 3.2 Sample methylation

[0127] After methylation, hydrolysis and acetylation, the samples were determined by GC-MS and compared with the standard mass spectrum library.

[0128] Weigh a polysaccharide sample (2-3 mg) into a glass reaction vial. Add 1 mL of anhydrous DMSO and quickly add methylation reagent solution A. Seal the vial, dissolve under ultrasound, and then add methylation reagent solution B. React in a magnetically stirred water bath at 30°C for 60 minutes. Finally, add 2 mL of ultrapure water to terminate the methylation reaction.

[0129] Take the methylated polysaccharide and add 1 ml of 2 M trifluoroacetic acid (TFA) for hydrolysis for 90 minutes, then evaporate to dryness on a rotary evaporator. Add 2 ml of double-distilled water and 60 mg of sodium borohydride to the residue for reduction for 8 hours, neutralize with glacial acetic acid, rotary evaporate, and oven dry at 101°C. Then, add 1 ml of acetic anhydride for acetylation at 100°C for 1 hour, then cool. Then, add 3 ml of toluene, concentrate under reduced pressure, and evaporate to dryness. Repeat this process 4-5 times to remove excess acetic anhydride.

[0130] The acetylated product was dissolved in 3 mL of CH₂Cl₂ and transferred to a separatory funnel. A small amount of distilled water was added and shaken thoroughly, followed by removal of the upper aqueous layer. This process was repeated four times. The CH₂Cl₂ layer was dried over an appropriate amount of anhydrous sodium sulfate, brought to a volume of 10 mL, and placed in a liquid phase vial. The acetylated product samples were analyzed using a Shimadzu GCMS-QP 2010 gas chromatograph-mass spectrometer.

[0131] GC-MS conditions: RXI-5SIL MS column 30m*0.25mm*0.25um; temperature program conditions: starting temperature 120℃, heating to 250℃ / min at 3℃ / min; hold for 5min; injection port temperature 250℃, detector temperature 250℃, carrier gas helium, flow rate 1mL / min.

[0132] 4. Experimental Results

[0133] The GCMS chromatogram of the sample (PMAA) is shown in Figure 9.

[0134] The results of polysaccharide methylated alditol acetyl ester (PMAA) are shown in the table below and in Figures 10-15.

[0135] 4. NMR analysis spectrum and analysis

[0136] 1. Experimental Materials and Instruments

[0137] Heavy water (D2O, 99.9%) and deuterated acetone were used as internal references; freeze dryer and nuclear magnetic resonance (NMR) instrument were Bruker 600M.

[0138] 2. Experimental Procedure

[0139] Weigh 50 mg of polysaccharide sample, dissolve it in 0.5 ml of heavy water and freeze-dry it. Then, dissolve the freeze-dried powder in 0.5 ml of heavy water and continue freeze-drying. Repeat the above process to fully exchange the active hydrogen. Then dissolve the sample in 0.5 ml of heavy water and place it in a 600 MHz nuclear magnetic resonance instrument at room temperature 25 ° C. 1 H NMR spectrum, 13 C NMR spectrum, DEPT135 one-dimensional spectrum and two-dimensional spectrum.

[0140] 3. Experimental Results

[0141] The hydrogen spectrum signals are mainly concentrated between 3.0 and 5.5 ppm. δ3.2-4.0 ppm is the sugar ring proton signal, and the main terminal proton peaks are δ4.42, 4.45, 4.49, 4.50, 4.66, and 4.71, with the signal peaks concentrated in the 4.3-5.5 ppm region, as shown in Figure 16.

[0142] Carbon spectrum analysis 13 C NMR (201 MHz, D2O): The C NMR spectrum signals are mainly concentrated between 60-120 ppm. By observing the C NMR spectrum, we can see that the main anomeric carbon signal peaks are δ103.82, 103.89, 104.02, 104.02, 104.12, and 104.21. The anomeric carbon region is mainly between δ93 and 105. The δ values ​​are 72.72, 74.55, 69.23, 76.08, 60.7, 76.69, 74.09, 77.24, 75.40, 68.82, 74.16, 71.57, 80.15, 76.68, 61.66, 74.76, 84.64, 69.63, 77.03, 62.04, 74.54, 83.90, 71.04, 76.10, 70.28, 74.52, 76.74, 76.41, 70.89, and 68.79 ppm. Based on the monosaccharide composition results, the polysaccharide is composed of glucose, indicating that the polysaccharide is mainly dextran, as shown in Figure 17.

[0143] Dept135 spectrum analysis shows that the peaks at 60.70, 68.82, 61.66, 62.04, 70.28, and 68.79 ppm are inverted peaks, indicating chemical shifts of C6, as shown in Figure 18.

[0144] As shown in Figures 19-22, the anomeric carbon signal is observed to be δ103.94 through HSQC spectra, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ4.71. Through HH-COSY, the signals of H1-2 are 4.71 / 3.44; the signals of H2-3 are 3.44 / 3.66; and the signals of H3-4 are 3.66 / 3.40. We can infer that H1, H2, H3, and H4 are δ4.71, 3.44, 3.66, and 3.40, respectively. The corresponding δ104.04, 74.55, 85.9, and 76.63. The corresponding C5 is 77.41; the chemical shift of C6 is δ62.04. Therefore, this signal should be attributed to the glycosidic bond →3)-β-Glcp-(1→

[0145] Through the HSQC spectrum, it can be observed that the anomeric carbon signal is δ104.21, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ4.42. Through HH-COSY, the signals of H1-2 are 4.42 / 3.23; the signals of H2-3 are 3.23 / 3.39; the signals of H3-4 are 3.39 / 3.55; we can infer that H1, H2, H3, and H4 are δ4.42, 3.23, 3.39, and 3.55, respectively, and their corresponding C1-4 are δ104.21, 74.52, 76.74, and 76.41; and the NOESY spectrum observes that δ4.43 has correlated peaks with 3.39, 3.55, 3.75, and 4.11. Dept135 combined with HSQC can determine that δ3.75 and 4.11 are attributed to the peaks of H6a, b, and H5 is 3.31ppm. The corresponding C5 is 70.89; the chemical shift of C6 is δ70.19, and the corresponding H6a is δ3.75, 4.11. Therefore, this signal should be attributed to the glycosidic bond →6)-β-Glcp-(1→

[0146] Based on similar rules and combined with HMBC and NOESY, all glycosidic bond signals were assigned as shown in the following table:

[0147] Hydrogen and carbon signal assignment

[0148] Main chain analysis:

[0149] In the HMBC spectrum, we attributed the glycosidic bond signals of the polysaccharide based on the one-dimensional and two-dimensional NMR spectra; the anomeric hydrogen of the glycosidic bond →6)-β-D-Glcp-(1→ and its own C6 have related signal peaks, indicating the existence of a linking mode of →6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→.

[0150] The anomeric hydrogen of the glycosidic bond →6)-β-D-Glcp-(1→ and C6 of its →3,6)-β-D-Glcp-(1→) have related signal peaks, indicating the existence of a linking mode of →6)-β-D-Glcp-(1→3,6)-β-D-Glcp-(1→.

[0151] The anomeric hydrogen of the glycosidic bond →3,6)-β-D-Glcp-(1→ and C6 of its →4,6)-β-D-Glcp-(1→) have related signal peaks, indicating the existence of the →3,6)-β-D-Glcp-(1→4,6)-β-D-Glcp-(1→ linking mode.

[0152] Branch chain analysis:

[0153] The anomeric carbon of the glycosidic bond →3)-β-D-Glcp-(1→ and its H3 of →3,6)-β-D-Glcp-(1→ have related signal peaks, indicating the existence of the linking mode of →3)-β-D-Glcp-(1→3,6)-β-D-Glcp-(1→.

[0154] In the NOESY spectrum,

[0155] The anomeric hydrogen of the glycosidic bond β-D-Glcp-(1→) and H4 of its →4)-β-D-Glcp-(1→) have related signal peaks, indicating the existence of the β-D-Glcp-(1→4)-β-D-Glcp-(1→ linking mode.

[0156] The anomeric hydrogen of the glycosidic bond β-D-Glcp-(1→) and H3 of its →3)-β-D-Glcp-(1→) have related signal peaks, indicating the existence of a linking mode of β-D-Glcp-(1→3)-β-D-Glcp-(1→.

[0157] The anomeric hydrogen of the glycosidic bond →4)-β-D-Glcp-(1→ and its H4 of →4,6)-β-D-Glcp-(1→ have related signal peaks, indicating the existence of the linking mode of →4)-β-D-Glcp-(1→4,6)-β-D-Glcp-(1→.

[0158] In summary, we can infer that the main chain of this polysaccharide is β-1,6 glucan, and β-D-Glcp-(1→3)-β-D-Glcp-(1→ and β-D-Glcp-(1→4)-β-D-Glcp-(1→) are connected to the main chain through the o-3 bond of →3,6)-β-D-Glcp-(1→) and the o-4 bond of →4,6)-β-D-Glcp-(1→, respectively. The simplified molecular structure formula is shown below.

[0159] Another object of the present invention is to provide an application of GLP-3, a polysaccharide of Ganoderma lucidum. The polysaccharide GLP-3 of Ganoderma lucidum has good water solubility, is easily absorbed by the human body, has anti-tumor efficacy and is effective in preventing the occurrence of tumors in humans. Especially when used in combination with chemotherapy drugs, it can eliminate the toxic side effects of chemotherapy drugs on the human body and is effective in controlling and reducing tumor masses and reducing and eliminating tumor cells.

[0160] The Ganoderma lucidum polysaccharide GLP-3 is used in drugs for inhibiting tumor spread or enhancing human immunity.

[0161] Experimental study and data on the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on LLC lung cancer-bearing mice

[0162] Purpose of the experiment

[0163] Tumor-bearing mice were prepared by subcutaneously inoculating LLC lung cancer tumor homogenate in the right axilla of C57 mice to study the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-3 on lung cancer tumor-bearing mice and provide experimental basis for its clinical research.

[0164] Experimental Materials

[0165] Test sample

[0166] Ganoderma lucidum polysaccharide GLP-3 was provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0167] Positive control

[0168] Cisplatin, batch number: E2128081, product of Shanghai Aladdin Biochemical Technology Co., Ltd.

[0169] experimental animals

[0170] Seventy-five SPF male C57 mice, weighing 14–16 g, were provided by the Guangdong Medical Laboratory Animal Center. The laboratory animal production license number is SCXK (Yue) 2022-0002, and the laboratory animal quality certificate number is 44007200103827.

[0171] Main reagents

[0172] PBS buffer was prepared by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.; fetal bovine serum was a product of Zhejiang Tianhang Biotechnology Co., Ltd.; DMEM culture medium was a product of Gibco; and 0.25% trypsin was a product of Gibco.

[0173] Main instruments

[0174] Vernier caliper, product of Shanghai Tool Factory Co., Ltd.; I-2000 balance, product of Dongguan Nancheng Changxie Electronic Products Factory; ophthalmic scissors and tweezers, product of Shanghai Jinzhong Medical Instrument Co., Ltd.; CCL-170B-8 carbon dioxide incubator, product of Singapore ESCO; Luna-Ⅱ cell counter, product of Nanjing Hengqiao Instrument Co., Ltd.; JRA-35S handheld homogenizer, product of Wuxi Jerian Instrument Equipment Co., Ltd.

[0175] Experimental methods

[0176] Normally grown LLC cells were inoculated subcutaneously on the left shoulder of 5 healthy male C57 mice. 3 When the tumor mass was removed and made into a homogenate suspension, it was injected into the axillary subcutaneous tissue of 60 healthy male C57 mice to establish a solid tumor model. 3 At approximately 60 days, mice were randomly divided into groups based on tumor volume and given the corresponding drug or vehicle by gavage or intraperitoneal injection for 19 consecutive days. Tumor diameters were measured every 3 days to calculate tumor volume, and mice were weighed every 3 days. At the end of the experiment, tumors, spleens, and thymuses were removed and weighed, and the tumor index, spleen index, and thymus index were calculated.

[0177] Dosage design

[0178] Ganoderma lucidum polysaccharide GLP-3 was designed based on previous experimental results. The low dose was 50 mg / kg and the high dose was 150 mg / kg. The corresponding drugs were administered as shown in Table 1.

[0179] Cisplatin dosage design basis: According to the clinical dosage of cisplatin, each person should not exceed 100 mg / m2 per day. 2 The tolerance of mice to cisplatin was evaluated, and a dose of 4 mg / kg was selected as the administration dose.

[0180] Table 1 Trial grouping and dosage design

[0181] Detection indicators

[0182] Efficacy indicators

[0183] Relative tumor inhibition rate

[0184] Relative tumor inhibition rate (%) = (1-T RTV / C RTV )×100%. Among them, T RTV is the relative tumor volume of the experimental group, C RTV is the relative tumor volume of the model control group. t / V0,V t=V0 is the tumor volume of mice on day t of drug administration, and V0 is the tumor volume of mice at the time of grouping. Evaluation criteria: Relative tumor inhibition rate ≥ 40%, and P < 0.05 after statistical analysis is considered an effective inhibition level.

[0185] Tumor growth inhibition rate

[0186] Tumor growth inhibition rate (%) = (1-T / C) × 100%. Where T represents the average tumor weight of the treatment group and C represents the average tumor weight of the model control group. Evaluation criteria: Tumor growth inhibition rate ≥ 40% and statistical analysis P < 0.05 are considered effective inhibition levels.

[0187] Spleen and thymus organ coefficients: After the last administration, the spleen, thymus, and tumor were weighed and the organ coefficients were calculated.

[0188] Tumor index (%) = tumor weight / body weight × 100%.

[0189] Immune organ index (mg / g) = organ mass / body weight × 1000.

[0190] Data processing and statistical analysis

[0191] SPSS 17.0 was used for statistical analysis, and the statistical significance level was set at P ≤ 0.05. The measurement data were expressed as mean ± standard deviation. Leven's test was used to test normality and homogeneity of variance. If normality and homogeneity of variance were met (P>0.05), one-way analysis of variance (ANOVA) and LSD test were used for statistical analysis. If normality and homogeneity of variance were not met (P<0.05), Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P<0.05), Dunnett's Test (non-parametric method) was used for comparative analysis. Statistical differences and biological significance were considered in the evaluation.

[0192] Experimental results

[0193] Animal deaths

[0194] As shown in Table 2, the mortality rate of mice in each group in this experiment was 0.

[0195] Table 2 Statistics of the number of surviving animals and mortality rates in each group

[0196] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on body weight in LLC lung cancer-bearing mice

[0197] As shown in Table 3, compared with the normal group, the body weight of mice in the model control group was significantly increased on D0 and D9-D19, the body weight of mice in the cisplatin group and the high-dose cisplatin + GLP-3 group was significantly decreased on D0 and D6-D19, and the body weight of mice in the low-dose cisplatin + GLP-3 group was significantly decreased on D6-D19.

[0198] Compared with the model control group, the body weight of mice in the cisplatin group and the high-dose cisplatin + GLP-3 group was significantly reduced from D6 to D19, and the body weight of mice in the low-dose cisplatin + GLP-3 group was significantly reduced from D3 to D19.

[0199] Compared with the cisplatin group, the body weight of mice in the cisplatin + GLP-3 low-dose group was significantly reduced from D12 to D19.

[0200] Table 3 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on body weight of LLC lung cancer-bearing mice

[0201] Note: Compared with the model control group, + P<0.05; compared with the cisplatin group, # P<0.05; compared with the normal group, * P<0.05.

[0202] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on tumor volume in LLC lung cancer-bearing mice

[0203] As shown in Table 4, compared with the model control group, the tumor volume of the cisplatin group was significantly reduced from D9 to D19, and the tumor volume of the cisplatin + GLP-3 low-dose group and the cisplatin + GLP-3 high-dose group was significantly reduced from D6 to D19.

[0204] Compared with the cisplatin group, the tumor volume of the low-dose cisplatin + GLP-3 group was significantly reduced from D9 to D19, and the tumor volume of the high-dose cisplatin + GLP-3 group was significantly reduced from D12 to D19.

[0205] Table 4 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on tumor volume in LLC lung cancer-bearing mice

[0206] Note: Compared with the model control group, + P<0.05; compared with the cisplatin group, # P<0.05.

[0207] Effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the relative tumor inhibition rate of LLC lung cancer-bearing mice

[0208] As shown in Tables 5 and 6, compared with the model control group, the relative tumor inhibition rates of the cisplatin group were above 40% on D9 to D19, which were 54.6%, 63.8%, 77.3% and 79.5% respectively; the relative tumor inhibition rates of the cisplatin + GLP-3 low-dose group were above 50% on D6 to D19, which were 51.9%, 70.0%, 80.1%, 87.4% and 92.2% respectively; the relative tumor inhibition rates of the cisplatin + GLP-3 high-dose group were above 40% on D6 to D19, which were 43.3%, 67.1%, 74.9%, 82.3% and 88.0% respectively.

[0209] Compared with the cisplatin group, the relative tumor inhibition rates of the low-dose cisplatin + GLP-3 group were 44.9%, 44.6%, and 62.3% on D12 to D19 (P<0.05), and the relative tumor inhibition rates of the high-dose cisplatin + GLP-3 group were 30.6%, 22.3%, and 41.6% on D12 to D19 (P<0.05).

[0210] Table 5 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the relative tumor inhibition rate of LLC lung cancer-bearing mice (compared with the model control group)

[0211] Note: Compared with the model control group, + P<0.05.

[0212] Table 6 Effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the relative tumor inhibition rate of LLC lung cancer-bearing mice (compared with the cisplatin group)

[0213] Note: Compared with cisplatin group, # P<0.05.

[0214] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on organ index and tumor growth inhibition rate in LLC lung cancer-bearing mice

[0215] As shown in Table 7, compared with the model group, the tumor index, spleen index, and thymus index were significantly decreased in the cisplatin group, the low-dose cisplatin + GLP-3 group, and the high-dose cisplatin + GLP-3 group. Compared with the cisplatin group, the tumor index was significantly decreased in the low-dose cisplatin + GLP-3 group. Compared with the normal group, the spleen index was significantly increased in the model control group and significantly decreased in the low-dose cisplatin + GLP-3 group. The thymus index was significantly decreased in the model control group, the cisplatin group, the low-dose cisplatin + GLP-3 group, and the high-dose cisplatin + GLP-3 group.

[0216] Compared with the model control group, the tumor growth inhibition rates in the cisplatin group were 74.4%, 91.7% in the low-dose cisplatin + GLP-3 group, and 84.0% in the high-dose cisplatin + GLP-3 group. Compared with the cisplatin group, the tumor growth inhibition rates in the low-dose cisplatin + GLP-3 group and the high-dose cisplatin + GLP-3 group were 67.4% and 37.4%, respectively.

[0217] Table 7 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on organ index and tumor growth inhibition rate in LLC lung cancer-bearing mice

[0218] Note: Compared with the model control group, + P<0.05; compared with the cisplatin group, # P<0.05; compared with the normal group, * P<0.05.

[0219] Figures 23, 24, 25, and 26 show images of tumor-bearing mice, corresponding to the following groups: Figure 23: Model control group, Figure 24: Cisplatin group, Figure 25: Cisplatin + GLP-3 low-dose group, Figure 26: Cisplatin + GLP-3 high-dose group

[0220] As shown in Figures 27, 28, 29 and 30, they are pictures of tumors in tumor-bearing mice, and the corresponding groups are: Figure 27: model control group, Figure 28: cisplatin group, Figure 29: cisplatin + GLP-3 low-dose group, and Figure 30: cisplatin + GLP-3 high-dose group.

[0221] in conclusion

[0222] Ganoderma lucidum polysaccharide GLP-3 combined with cisplatin can significantly inhibit the growth of tumors in LLC lung cancer-bearing mice and has a significant synergistic effect.

[0223] Experimental study and data on the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on H22 liver cancer-bearing mice

[0224] Purpose of the experiment

[0225] The H22 liver cancer homogenate was inoculated into the right axilla of C57 mice to prepare tumor-bearing mice. The effect of Ganoderma lucidum polysaccharide GLP-3 on liver cancer-bearing mice was studied to provide experimental basis for its clinical research.

[0226] Experimental Materials

[0227] Test sample

[0228] Ganoderma lucidum polysaccharide GLP-3 was provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0229] Positive control

[0230] Cisplatin, batch number: E2128081, product of Shanghai Aladdin Biochemical Technology Co., Ltd.

[0231] experimental animals

[0232] Sixty-five SPF male C57 mice, weighing 16–18 g, were provided by the Guangdong Medical Laboratory Animal Center. The laboratory animal production license number is SCXK (Yue) 2022-0002, and the laboratory animal quality certificate number is 44007200103599.

[0233] Main reagents

[0234] PBS buffer was prepared by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.; fetal bovine serum was a product of Zhejiang Tianhang Biotechnology Co., Ltd.; 1640 culture medium was a product of Gibco; and 0.25% trypsin was a product of Gibco.

[0235] Main instruments

[0236] Vernier caliper, product of Shanghai Tool Factory Co., Ltd.; I-2000 balance, product of Dongguan Nancheng Changxie Electronic Products Factory; ophthalmic scissors and tweezers, product of Shanghai Jinzhong Medical Instrument Co., Ltd.; CCL-170B-8 carbon dioxide incubator, product of Singapore ESCO; Luna-Ⅱ cell counter, product of Nanjing Hengqiao Instrument Co., Ltd.; JRA-35S handheld homogenizer, product of Wuxi Jerian Instrument Equipment Co., Ltd.

[0237] Experimental methods

[0238] The H22 liver cancer solid tumor homogenate suspension was injected subcutaneously into the axilla of 50 healthy male C57 mice to establish a solid tumor model. 3 At approximately 60 days, mice were randomly divided into groups based on tumor volume and given the corresponding drug or vehicle by gavage or intraperitoneal injection for 22 consecutive days. Tumor diameters were measured every 3 days to calculate tumor volume, and mice were weighed every 3 days. At the end of the experiment, tumors, spleens, and thymuses were removed and weighed, and the tumor index, spleen index, and thymus index were calculated.

[0239] Dosage design

[0240] Ganoderma lucidum polysaccharide GLP-3 was designed based on previous experimental results. The low dose was 50 mg / kg and the high dose was 150 mg / kg. The corresponding drugs were administered as shown in Table 8.

[0241] Cisplatin dosage design basis: According to the clinical dosage of cisplatin, each person should not exceed 100 mg / m2 per day. 2 The tolerance of mice to cisplatin was evaluated and a dose of 3 mg / kg was selected as the administration dose.

[0242] Table 8 Trial grouping and dosage design

[0243] Detection indicators

[0244] Efficacy indicators

[0245] Relative tumor inhibition rate

[0246] Relative tumor inhibition rate (%) = (1-T RTV / C RTV )×100%. Among them, T RTV is the relative tumor volume of the experimental group, C RTV is the relative tumor volume of the model control group. t / V0,V t =V0 is the tumor volume of mice on day t of drug administration, and V0 is the tumor volume of mice at the time of grouping. Evaluation criteria: Relative tumor inhibition rate ≥ 40%, and P < 0.05 after statistical analysis is considered an effective inhibition level.

[0247] Tumor growth inhibition rate

[0248] Tumor growth inhibition rate (%) = (1-T / C) × 100%. Where T represents the average tumor weight of the treatment group and C represents the average tumor weight of the model control group. Evaluation criteria: Tumor growth inhibition rate ≥ 40% and statistical analysis P < 0.05 are considered effective inhibition levels.

[0249] Spleen and thymus organ coefficients: After the last administration, the spleen, thymus, and tumor were weighed and the organ coefficients were calculated.

[0250] Tumor index (%) = tumor weight / body weight × 100%.

[0251] Immune organ index (mg / g) = organ mass / body weight × 1000.

[0252] Data processing and statistical analysis

[0253] SPSS 17.0 was used for statistical analysis, and the statistical significance level was set at P ≤ 0.05. The measurement data were expressed as mean ± standard deviation. Leven's test was used to test normality and homogeneity of variance. If normality and homogeneity of variance were met (P>0.05), one-way analysis of variance (ANOVA) and LSD test were used for statistical analysis. If normality and homogeneity of variance were not met (P<0.05), Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P<0.05), Dunnett's Test (non-parametric method) was used for comparative analysis. Statistical differences and biological significance were considered in the evaluation.

[0254] Experimental results

[0255] Animal deaths

[0256] As shown in Table 9, the mortality rate of mice in the model control group was 50%, and the mortality rate of mice in the other groups was 0.

[0257] Table 9 Statistics of the number of surviving animals and mortality rates in each group

[0258] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on body weight in H22 liver cancer-bearing mice

[0259] As shown in Table 10, compared with the model control group, the body weight of mice in the cisplatin group was significantly reduced on D6 and D12-D22, the body weight of mice in the low-dose cisplatin + GLP-3 group was significantly reduced on D6-D22, and the body weight of mice in the high-dose cisplatin + GLP-3 group was significantly reduced on D3-D22.

[0260] Compared with the cisplatin group, the body weight of mice in the low-dose cisplatin + GLP-3 group was significantly reduced on D3, D6, and D12 to D22, and the body weight of mice in the high-dose cisplatin + GLP-3 group was significantly reduced on D0 to D22.

[0261] Table 10 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on body weight of H22 liver cancer-bearing mice

[0262] Note: Compared with the model control group, + P<0.05; compared with the cisplatin group, # P<0.05; compared with the normal group, * P<0.05.

[0263] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on tumor volume in H22 liver cancer-bearing mice

[0264] As shown in Table 11, compared with the model control group, the tumor volume of the cisplatin group was significantly reduced from D9 to D22, and the tumor volume of the cisplatin + GLP-3 low-dose group and the cisplatin + GLP-3 high-dose group was significantly reduced on D3 and D9 to D22.

[0265] Compared with the cisplatin group, the tumor volume of the low-dose and high-dose cisplatin + GLP-3 groups was significantly reduced from D9 to D22.

[0266] Table 11 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on tumor volume in H22 liver cancer-bearing mice

[0267] Note: Compared with the model control group, +P<0.05; compared with the cisplatin group, # P<0.05.

[0268] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on relative tumor inhibition rate in H22 liver cancer-bearing mice

[0269] As shown in Tables 12 and 13, compared with the model control group, the relative tumor inhibition rates of the cisplatin group were above 50% on D9 to D22, namely 53.1%, 64.4%, 73.1%, 71.6% and 75.0%, respectively; the relative tumor inhibition rates of the cisplatin + GLP-3 low-dose group were above 70% on D9 to D22, namely 72.1%, 75.9%, 84.2%, 81.7% and 80.4%, respectively; the relative tumor inhibition rates of the cisplatin + GLP-3 high-dose group were above 75% on D9 to D22, namely 77.6%, 83.4%, 89.2%, 90.4% and 88.0%, respectively.

[0270] Compared with the cisplatin group, the relative tumor inhibition rates of the low-dose cisplatin + GLP-3 group were 40.5%, 32.3%, 41.2%, 35.7%, and 21.4% on D9-D22 (P<0.05). The relative tumor inhibition rates of the high-dose cisplatin + GLP-3 group were above 50% on D9-D22, namely 52.3%, 53.3%, 59.9%, 66.2%, and 51.9% (P<0.05).

[0271] Table 12 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the relative tumor inhibition rate of H22 liver cancer-bearing mice (compared with the model control group)

[0272] Note: Compared with the model control group, + P<0.05.

[0273] Table 13 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the relative tumor inhibition rate of H22 liver cancer-bearing mice (compared with the cisplatin group)

[0274] Note: Compared with cisplatin group, # P<0.05.

[0275] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on organ indexes and tumor growth inhibition rate in H22 liver cancer-bearing mice

[0276] As shown in Table 14, compared with the model group, the tumor index, spleen index, and thymus index were significantly decreased in the cisplatin group, the low-dose cisplatin + GLP-3 group, and the high-dose cisplatin + GLP-3 group. Compared with the cisplatin group, the tumor index was significantly decreased in the high-dose cisplatin + GLP-3 group, and the spleen index was significantly decreased in the low-dose cisplatin + GLP-3 group and the high-dose cisplatin + GLP-3 group. Compared with the normal group, the spleen index in the model control group was significantly increased, while the thymus index in the cisplatin group, the low-dose cisplatin + GLP-3 group, and the high-dose cisplatin + GLP-3 group was significantly decreased.

[0277] Compared with the model control group, the tumor growth inhibition rates in the cisplatin group were 75.7%, 82.8% in the low-dose cisplatin + GLP-3 group, and 85.0% in the high-dose cisplatin + GLP-3 group. Compared with the cisplatin group, the tumor growth inhibition rates in the low-dose cisplatin + GLP-3 group and the high-dose cisplatin + GLP-3 group were 29.4% and 38.4%, respectively.

[0278] Table 14 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on organ index and tumor growth inhibition rate in H22 liver cancer-bearing mice

[0279] Note: Compared with the model control group, + P<0.05; compared with the cisplatin group, # P<0.05; compared with the normal group, * P<0.05.

[0280] As shown in Figures 31-34, these are pictures of tumor-bearing mice, and the corresponding groups are: Figure 31: Model control group, Figure 32: Cisplatin group, Figure 33: Cisplatin + GLP-3 low-dose group, Figure 34: Cisplatin + GLP-3 high-dose group

[0281] Figures 35-38 show tumor images of tumor-bearing mice, and the corresponding groups are: Figure 35: Model control group, Figure 36: Cisplatin group, Figure 37: Cisplatin + GLP-3 low-dose group, Figure 38: Cisplatin + GLP-3 high-dose group

[0282] in conclusion

[0283] Ganoderma lucidum polysaccharide GLP-3 combined with cisplatin can significantly inhibit the growth of tumors in H22 liver cancer-bearing mice and has a significant synergistic effect.

[0284] Experimental study and data on the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on H22 orthotopic liver cancer in mice

[0285] Purpose of the experiment

[0286] In this study, H22 liver cancer cells were inoculated in the right axilla of C57 mice to prepare mice with orthotopic tumors. The anti-tumor effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on H22 orthotopic liver cancer mice was studied to provide experimental basis for its clinical research.

[0287] Experimental Materials

[0288] Test sample

[0289] Ganoderma lucidum polysaccharide GLP-3, batch number: ALM20200518A1, was provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0290] Positive control

[0291] Kanglaite soft capsules, batch number: 20211009, Zhejiang Kanglaite Pharmaceutical Co., Ltd.; cisplatin injection, batch number: 601211204, Jiangsu Hausen Pharmaceutical Group Co., Ltd.

[0292] experimental animals

[0293] One hundred SPF male C57 mice, weighing 12-15 g, were provided by Hunan Slake Jingda Experimental Animal Co., Ltd., with an experimental animal production license number of SCXK (Xiang) 2019-0004 and an experimental animal quality certificate number of No. 430727221101898084 and No. 430727221101898112. They were raised in Area D of the barrier environment animal laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd. with an experimental animal use license number of SYXK (Xiang) 2020-0015.

[0294] Main reagents

[0295] 0.9% sodium chloride injection, batch number: 21071401C, product of Hunan Kangyuan Pharmaceutical Co., Ltd.; veterinary blood cell analysis diluent, batch number: 2022052603, veterinary ALT assay kit, batch number: 201751, AST assay kit, batch number: 201750, CRE assay kit, batch number: 111644, BUN assay kit, batch number: 201749, all are products of Wako Pure Chemical Industries, Ltd. of Japan.

[0296] Main instruments

[0297] AR223CN electronic balance, Ohaus Instrument (Changzhou) Co., Ltd.; LABOSPECT003 automatic biochemical analyzer, Hitachi, Japan; AniView100 multi-mode animal live imaging system, ANDOR; TDZ5-WS desktop multi-tube automatic balancing centrifuge, Hunan Keda Industrial Development Co., Ltd.; ME2002E electronic balance, Shimadzu Corporation, Japan; flow cytometer, BD; ASP200S fully automatic tissue dehydrator, ASP300S fully automatic tissue dehydrator, TP1020 fully automatic dehydrator, HI1210 slide spreader, HI1220 slide baking machine, RM2235 paraffin sectioner, EG1150H+C tissue embedding machine, AutoStainer XL automatic slide stainer + CV5030 automatic coverslipper, BX43 biological microscope + MD50 digital imaging system, CX31 biological microscope, Leica, Germany.

[0298] Experimental methods

[0299] The concentration of Luc-labeled fluorescence was 1×10 7 Hepatocellular carcinoma (H22) cells at a concentration of 1 × 10 cells / mL were first inoculated into the peritoneal cavity of 8 male C57 mice. When the mice developed ascites, the ascites was aspirated aseptically, washed with HBSS buffer, centrifuged and the supernatant discarded, stained with trypan blue, and the ascites cells were counted under a microscope. The cell count was adjusted to 1 × 10 cells / mL with HBSS buffer. 13 80 male C57 mice were inoculated with 10 μL of cisplatin per mL into the right liver region to create orthotopic tumor-bearing mice. One week later, liver tumor formation in the mice was assessed using a small animal in vivo imaging device. The mice were randomly divided into the following groups according to tumor size: model control group, radiotherapy group (2 Gy / day), radiotherapy + cisplatin group (2 Gy + 4 mg / kg), Kanglaite soft capsule group (1404 mg / kg), radiotherapy + Kanglaite soft capsule group (2 Gy + 1404 mg / kg), radiotherapy + low-dose GLP-3 group (2 Gy + 130 mg / kg), and radiotherapy + high-dose GLP-3 group (2 Gy + 1170 mg / kg). Ten mice were included as a normal control group. Except for the normal control group, all other animals underwent radiotherapy using an animal radiotherapy device. The animals were anesthetized, placed in a homemade lead suit, and exposed to the tumor tissue for 5 consecutive days. The radiation intensity was 2 Gy / day for 5 consecutive days. The normal control group and the model control group were given pure water by gavage, the mice in the radiotherapy + cisplatin group were given cisplatin by intraperitoneal injection, and the mice in the other groups were given the corresponding drug solution, with the dosage volume being 20 mL / kg (gavage) and 10 mL / kg (intraperitoneal injection), once a day for 14 consecutive days. After the last dose, blood was collected from the orbit to detect blood WBC, RBC, liver and kidney function indicators (ALT, AST, BUN, CRE) and CD3 + / CD4 + 、CD3 + / CD8 + The spleen, thymus, and tumor were weighed and the organ coefficients were calculated. The liver, spleen, and thymus were subjected to histopathological examination.

[0300] Dosage design

[0301] Ganoderma lucidum polysaccharide GLP-3 was designed based on previous experimental results. The low dose was 130 mg / kg and the high dose was 1170 mg / kg. The corresponding drugs were administered as shown in Table 15.

[0302] The proposed clinical dosage of Kanglaite Soft Capsules is 0.45g / capsule, 6 capsules / time, 4 times / day, which is 10.8g / day. The equivalent dose in mice based on body surface area is 10.8g / day*0.0026 / 0.02kg=1404mg / kg. This trial was conducted at the same multiple of the proposed clinical dosage.

[0303] This study designed radiotherapy intensity to be 2 Gy / day, which is equal to the intended clinical dose.

[0304] Table 15 Trial grouping and dosage design

[0305] Detection indicators

[0306] Efficacy indicators

[0307] Animal survival rate and general condition: The animals were weighed once a week and the death of animals was recorded.

[0308] Tumor volume detection: Small animal in vivo imaging technology was used to detect the changes in tumor volume in each group every week.

[0309] Hematological examination: blood routine (WBC, RBC) and biochemical (liver and kidney function) indexes were tested after the last dose.

[0310] Immune organs: The thymus, spleen, tumor, and liver were weighed and the organ coefficient was calculated: Organ coefficient (%) = organ weight / fasting body weight × 100%.

[0311] CD4 + 、CD8 + Content detection: After the last administration, blood lymphocyte typing CD3 was detected by flow cytometry + / CD4 + , CD3 + / CD8 + content.

[0312] Data processing and statistical analysis

[0313] The significant figures of the experimental data were rounded off and statistical analysis was performed in accordance with the center's SOP. The statistical software used was SPSS. Normality and homogeneity of variance were tested using the Leven's test. If there was no statistical significance (P>0.05), one-way analysis of variance (ANOVA) was used for statistical analysis. If the ANOVA was statistically significant (P≤0.05), the LSD test (parametric method) was used for comparative analysis. If the variances were unequal (P≤0.05), the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P≤0.05), the Dunnett's test (nonparametric method) was used for comparative analysis. Statistical results were tested with an α=0.05 threshold, where P≤0.05 indicated statistical significance and P≤0.01 indicated that the difference being tested was highly significant.

[0314] Experimental results

[0315] Animal deaths

[0316] After administration, mice 2M07 / 2M08 and 2M05 in the model control group died on D4 and D10, respectively; mice 3M02 / 3M10, 3M07, 3M09 / 3M01 and 3M05 in the radiotherapy group died on D3, D4, D5 and D10, respectively; mice 4M03 / 4M09, 4M06, 4M10, 4M07 and 4M04 in the radiotherapy + cisplatin group died on D4, D6, D7, D8 and D13, respectively; mice 5M03, 5M07 and 5M05 in the Kanglaite soft capsule group died on D4, D6, D7, D8 and D13, respectively. 08 died on D8, D10 and D14 respectively; mice 6M10, 6M08 / 6M02, 6M05, 6M06, 6M01 / 6M07 in radiotherapy + Kanglaite soft capsule group died on D7, D8, D10, D13 and D14 respectively; mouse 7M01 in radiotherapy + low-dose GLP-3 group died on D8; mice 8M03, 8M08, 8M06, 8M02 in radiotherapy + high-dose GLP-3 group died on D9, D10, D13 and D14 respectively.

[0317] The mortality rates of each group were 0%, 30%, 60%, 60%, 30%, 70%, 10% and 50% respectively.

[0318] Table 16 Statistics of the number of surviving animals and mortality rates in each group

[0319] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on body weight in mice with H22 orthotopic hepatocellular carcinoma

[0320] As shown in Table 17, compared with the normal control group, the body weight of mice in the model control group was significantly decreased at 0, 1, and 2 weeks after administration (P ≤ 0.01). Compared with the model control group, the body weight of mice in the radiotherapy group, the radiotherapy combined with cisplatin group, the Kanglaite soft capsule group, and the low-dose GLP-3 group was significantly decreased at 1 and 2 weeks after administration (P ≤ 0.05 or P ≤ 0.01); the body weight of mice in the radiotherapy combined with high-dose GLP-3 group was significantly decreased at 1 week after administration (P ≤ 0.05). Compared with the radiotherapy group, the body weight of mice in the Kanglaite soft capsule group was significantly increased at 1 and 2 weeks after administration (P ≤ 0.05 or P ≤ 0.01). Compared with the radiotherapy + cisplatin group, the body weight of mice in the radiotherapy combined with low- and high-dose GLP-3 groups, and the Kanglaite soft capsule group was significantly increased at 1 and 2 weeks after administration (P ≤ 0.05 or P ≤ 0.01). There were no significant differences among the other treatment groups.

[0321] Table 17 Effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on body weight of mice bearing H22 orthotopic liver cancer (Mean ± SEM)

[0322] Note: Compared with the normal control group, ++ P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01; compared with the radiotherapy group, # P≤0.05, ## P≤0.01; compared with the radiotherapy + cisplatin group, & P≤0.05, && P≤0.01.

[0323] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on H22 orthotopic hepatocellular carcinoma in mice

[0324] As shown in Figures 39, 40, and Table 18, compared with the normal control group, the tumor counts of mice in the model control group increased significantly (P ≤ 0.01). Compared with the model control group, the tumor counts of mice in the radiotherapy combined with low- and high-dose GLP-3 groups, and the radiotherapy combined with cisplatin group decreased significantly after the second week of administration (P ≤ 0.05 or P ≤ 0.01). Compared with the Kanglaite soft capsule group, the tumor counts of mice in the radiotherapy combined with high-dose GLP-3 group decreased significantly after the second week of administration (P ≤ 0.05). There were no significant differences among the other groups.

[0325] As shown in Figure 39, the corresponding groups are: A: normal group, B: model control group, C: radiotherapy group, D: radiotherapy + cisplatin group, E: Kanglaite soft capsule group, F: radiotherapy + Kanglaite soft capsule group, G: radiotherapy + low-dose GLP-3 group, H: radiotherapy + high-dose GLP-3 group.

[0326] As shown in Figure 40, the corresponding groups are: 1: normal group, 2: model control group, 3: radiotherapy group, 4: radiotherapy + cisplatin group, 5: Kanglaite soft capsule group, 6: radiotherapy + Kanglaite soft capsule group, 7: radiotherapy + GLP-3 low-dose group, 8: radiotherapy + GLP-3 high-dose group.

[0327] Table 18 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on H22 orthotopic liver cancer in mice (Mean ± SEM)

[0328] Note: Compared with the normal control group, ++ P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01; compared with the radiotherapy group, # P≤0.05; compared with radiotherapy + cisplatin group, & P≤0.05; compared with Kanglaite soft capsule group, ★ P≤0.05; compared with radiotherapy + Kanglaite soft capsule group, ■ P≤0.05.

[0329] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on organ coefficients and tumor growth inhibition rate in mice with H22 orthotopic hepatocellular carcinoma

[0330] As shown in Table 19, compared with the normal control group, the spleen and liver organ coefficients of the model control group were significantly increased (P ≤ 0.01), and the thymus coefficient was significantly decreased (P ≤ 0.01). Compared with the model control group, the spleen coefficients of mice in the radiotherapy combined with low- and high-dose GLP-3 groups, the Kanglaite soft capsule group, and the cisplatin group were significantly decreased (P ≤ 0.05 or P ≤ 0.01). The liver coefficients of mice in the radiotherapy combined with low-dose GLP-3 and cisplatin groups were significantly decreased (P ≤ 0.05 or P ≤ 0.01). Compared with the radiotherapy group, the spleen coefficient of mice in the Kanglaite soft capsule group was significantly increased (P ≤ 0.05 or P ≤ 0.01). Compared with the radiotherapy + cisplatin group, the spleen coefficient of mice in the Kanglaite soft capsule group was significantly increased (P ≤ 0.05 or P ≤ 0.01). Compared with the Kanglaite soft capsule group, the spleen coefficient of mice in the radiotherapy combined with low- and high-dose GLP-3 groups was significantly decreased (P ≤ 0.01). Compared with the radiotherapy + Kanglaite soft capsule group, there was no significant difference among the groups.

[0331] Table 19 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on organ coefficients of H22 orthotopic liver cancer mice (Mean ± SEM)

[0332] Note: Compared with the normal control group, + P≤0.05, ++P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01; compared with the radiotherapy group, # P≤0.05; compared with radiotherapy + cisplatin group, & P≤0.05, && P≤0.01; compared with radiotherapy + Kanglaite soft capsule group, ■ P≤0.05.

[0333] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on blood biochemical parameters in mice with H22 orthotopic hepatocellular carcinoma

[0334] As shown in Table 20, compared with the normal control group, the blood WBC, AST, BUN, and CRE of mice in the model control group were significantly increased (P ≤ 0.05 or P ≤ 0.01). Compared with the model control group, the blood WBC, AST, BUN, and CRE of mice in the radiotherapy group were significantly decreased (P ≤ 0.05 or P ≤ 0.01) in the radiotherapy combined with low-dose GLP-3 group, the cisplatin group, and the Kanglaite soft capsule group; the blood WBC, BUN, and CRE of mice in the radiotherapy combined with high-dose GLP-3 group were significantly decreased (P ≤ 0.05 or P ≤ 0.01); and the blood WBC and CRE of mice in the Kanglaite soft capsule group were significantly decreased (P ≤ 0.05 or P ≤ 0.01). Compared with the radiotherapy group, the blood CRE levels of mice in the radiotherapy combined with low-dose and high-dose GLP-3 groups were significantly reduced (P ≤ 0.05). The blood WBC, AST, and BUN levels of mice in the Kanglaite soft capsule group were significantly increased (P ≤ 0.05 or P ≤ 0.01), and the CRE levels were significantly reduced (P ≤ 0.05). Compared with the radiotherapy + cisplatin group, the blood WBC, AST, and BUN levels of mice in the Kanglaite soft capsule group were significantly increased (P ≤ 0.05 or P ≤ 0.01). Compared with the Kanglaite soft capsule group, the blood AST and BUN levels of mice in the radiotherapy combined with low-dose GLP-3 group were significantly reduced (P ≤ 0.05 or P ≤ 0.01). The blood BUN levels of mice in the radiotherapy combined with high-dose GLP-3 and Kanglaite soft capsule groups were significantly reduced (P ≤ 0.05 or P ≤ 0.01). No significant differences were found among the other groups.

[0335] Table 20 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on blood biochemical indicators in mice with H22 orthotopic liver cancer (Mean ± SEM)

[0336] Note: Compared with the normal control group, + P≤0.05, ++ P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01; compared with the radiotherapy group, # P≤0.05, ## P≤0.01; compared with the radiotherapy + cisplatin group, &P≤0.05, && P≤0.01; compared with Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0337] Effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on blood CD3 in mice with H22 orthotopic hepatocellular carcinoma + / CD4 + 、CD3 + / CD8 + Impact

[0338] As shown in Table 21, compared with the normal control group, the blood CD3+ / CD4+ and CD3+ / CD8+ of mice in the model control group showed an upward trend, but no significant differences were found. Compared with the model control group, there were no significant differences in the drug-treated groups. Compared with the radiotherapy + cisplatin group, the radiotherapy combined with low-dose GLP-3 group and the Kanglaite soft capsule group, the blood CD3+ / CD8+ of mice in the Kanglaite soft capsule group was significantly reduced (P≤0.05 or P≤0.01). There were no significant differences in the other groups.

[0339] Table 21 Effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on blood CD3 in mice with H22 orthotopic liver cancer + / CD4 + 、CD3 + / CD8 + Effect (Mean±SEM)

[0340] Note: Compared with the model control group, *P≤0.05; compared with the radiotherapy + cisplatin group, & P≤0.05, && P≤0.01.

[0341] As shown in Figures 41, 42, and 43, mice in the model control group showed extensive H22 hepatocellular carcinoma cell infiltration and necrosis in the liver, an increase in the number of sinusoidal cells, and inflammatory cell infiltration. Extramedullary hematopoiesis in the spleen increased significantly, with diffuse red pulp infiltration. Thymus showed hepatocellular carcinoma cell infiltration, and the number of cortical and medullary lymphocytes decreased. Mice in the radiotherapy and radiotherapy + cisplatin groups showed extensive H22 hepatocellular carcinoma cell infiltration and necrosis in the liver, increased extramedullary hematopoiesis in the spleen's red pulp, decreased white pulp cell numbers, and decreased thymic lymphocyte numbers. Following the combined administration of GLP-3, a polysaccharide from Ganoderma lucidum, in all groups, liver hepatocellular carcinoma cell infiltration and necrosis decreased, extramedullary hematopoiesis in the spleen increased, the number of white pulp plasma cells increased, and the severity of thymic lesions was alleviated.

[0342] As shown in Figures 41, 42, and 43, the corresponding groups are: A: normal group, B: model control group, C: cisplatin group, D: Kanglaite soft capsule group, E: cisplatin + Kanglaite soft capsule group, F: cisplatin + GLP-3 low-dose group, G: cisplatin + GLP-3 high-dose group

[0343] in conclusion

[0344] Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy can significantly inhibit the growth of tumors in H22 orthotopic liver cancer mice and has a significant synergistic effect.

[0345] Discussion and Summary

[0346] Liver cancer is a malignant tumor with an extremely high mortality rate. Currently, surgical resection combined with chemotherapy and interventional treatment, as well as radiotherapy, only serve to alleviate symptoms, making a complete cure extremely difficult. Liver cancer is not sensitive to chemotherapy, especially in patients with advanced liver cancer. To date, there is no reliable evidence that systemic chemotherapy can improve overall survival in patients with advanced liver cancer.

[0347] The results of this experiment showed that the tumor volume of the mice in the model control group increased significantly, the spleen index and liver index increased significantly, the number of red blood cells in the blood increased significantly, the number of white blood cells decreased significantly, and the liver and kidney functions were significantly abnormal, indicating that the lymphatic system function of the mice in the model control group decreased during the tumorigenesis process. After the last administration, GLP-3 polysaccharide combined with radiotherapy can significantly inhibit the growth of mouse tumors, significantly reduce the thymus index and spleen index, significantly reduce the number of red blood cells in the blood of mice, and significantly reduce the content of liver and kidney function indicators. CD4 + T cells and CD8 + T cells mediate tumor immune responses, including CD8 + T cells are the main effector cells of tumor immunity. The results showed that GLP-3 combined with radiotherapy in the blood of mice with Ganoderma lucidum polysaccharide + / CD4 + 、CD3 + / CD8 + The content was significantly reduced, indicating that Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy can protect immune organs and enhance the body's autoimmune function, thereby reducing toxicity and improving anti-tumor effects. Histopathological results also showed that Ganoderma lucidum polysaccharide GLP-3 can enhance the body's immunity. In addition, compared with Kanglaite soft capsules combined with radiotherapy, the tumor, spleen, and thymus coefficients were significantly reduced in Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy. These experimental results show that the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy is stronger than that of Kanglaite soft capsules combined with radiotherapy.

[0348] In summary, Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy can significantly inhibit the growth of tumors in H22 orthotopic liver cancer mice and has a significant synergistic effect.

[0349] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Ganoderma lucidum polysaccharide GLP-3, characterized in that: The molecular structure of the Ganoderma lucidum polysaccharide GLP-3 Molecular formula: (C 66 H 110 O 55 ) n , where n=30-46.

2. The Ganoderma lucidum polysaccharide GLP-3 according to claim 1, characterized in that: n is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46.

3. A method for extracting GLP-3 from Ganoderma lucidum polysaccharide, characterized in that: The method comprises the following steps: S1, removing dust from the ganoderma lucidum, drying it, and then crushing it to make ganoderma lucidum powder; S2, placing the crushed ganoderma lucidum powder in a sealed container, mixing with water and heating, and fully blending the ganoderma lucidum powder and water into a medicinal juice solution under high temperature and high pressure; S3, using membrane concentration technology to separate the medicinal juice solution to obtain a concentrated solution with medicinal ingredients and incompletely fused medicinal residues; S4. The concentrated solution containing the active ingredient is mixed with pure water in a ratio to a preset concentration of aqueous solution, and the aqueous solution is subjected to multiple column chromatography separations and concentrated freeze-dried to obtain Ganoderma lucidum polysaccharide GLP-3 containing the active ingredient.

4. The method for extracting GLP-3 from Ganoderma lucidum polysaccharide according to claim 3, characterized in that: In step S2, the ganoderma powder and water are mixed and fully heated to 105-200° C., and the boiling time lasts for 2-6 hours. The ganoderma powder and water in the sealed container are fully fused into a mixed medicinal liquid under a high temperature and high pressure environment.

5. The method for extracting GLP-3 from Ganoderma lucidum polysaccharide according to claim 4, characterized in that: In step S2, the ganoderma powder and water are mixed and fully heated to 105-170° C., and the boiling time lasts for 3-6 hours. The ganoderma powder and water in the sealed container are fully fused into a mixed medicinal liquid under a high temperature and high pressure environment.

6. The method for extracting GLP-3 from Ganoderma lucidum polysaccharide according to claim 5, characterized in that: In step S4, the concentrated solution containing the medicinal ingredients and pure water are prepared into a concentration ratio of 1:2-1:

5.

7. The method for extracting GLP-3 from Ganoderma lucidum polysaccharide according to claim 6, characterized in that: In step S3, the aqueous solution containing the extracted medicinal ingredients is concentrated by using membrane concentration technology to remove the Ganoderma lucidum residues therein to obtain a concentrated solution or paste of medicinal ingredients.

8. The method for extracting GLP-3 from Ganoderma lucidum polysaccharide according to claim 7, characterized in that: In step S1, the ganoderma lucidum is washed with clean water to remove the dust on the surface, and then dried at a high temperature of 105° C. by a drying device. The dried ganoderma lucidum is placed in a crushing device for crushing to obtain ganoderma lucidum powder, wherein the ganoderma lucidum powder is larger than 60 meshes.

9. The method for extracting GLP-3 from Ganoderma lucidum polysaccharide according to claim 8, characterized in that: In step S2, the mixed liquid in the sealed container is heated to 105°C, 110°C, 115°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C or 170°C, and the boiling time is 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h. The Ganoderma lucidum powder and water in the sealed container are fully fused into a mixed medicinal liquid under a high temperature and high pressure environment.

10. The use of Ganoderma lucidum polysaccharide GLP-3 according to claim 1 or 2, characterized in that: The Ganoderma lucidum polysaccharide GLP-3 has good water solubility, is easily absorbed by the human body, has anti-tumor efficacy and has a powerful effect on preventing the occurrence of tumors in humans. Especially when used in combination with chemotherapy drugs, it can eliminate the toxic side effects of chemotherapy drugs on the human body and has an effect on controlling, reducing tumor masses and reducing and eliminating tumor cells.

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