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

Abstract

The present invention is applicable to the technical field of plant extraction and separation, and provides a polysaccharide compound with a clear molecular structure, having anti-tumor efficacy and capable of eliminating the toxic and side effects of chemotherapy. The polysaccharide compound is used for treating patients with advanced cancer (the surgical condition has been lost, the survival period is only three to six months, and the chemotherapy condition is still available). The molecular formula of the polysaccharide compound is: (C 66 H 110 O 55 ) n ; The polysaccharide with this active ingredient is obtained by extracting the active ingredients of Ganoderma lucidum under high temperature and high pressure to obtain the active ingredient polysaccharide. The polysaccharide with this active ingredient has good water solubility, is easily absorbed by the human body, has anti-tumor efficacy and has an effect on preventing the occurrence of tumors in humans. Especially when used in combination with chemotherapy drugs, it can eliminate the toxic and side effects caused by chemotherapy drugs on the human body, and there are corresponding experimental data to prove the control, reduction of tumor mass, and reduction and elimination of tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of improvement of plant extraction and separation technologies, and particularly relates to a method for extracting a polysaccharide compound with a clear molecular structure, having anti-tumor efficacy and capable of eliminating the toxic and side effects of chemotherapy drugs, namely Ganoderma lucidum polysaccharide GLP-3, and its uses. Background Art

[0002] Ganoderma lucidum is a type of fungal plant with a long medicinal history in China and Japan. The active ingredients in Ganoderma lucidum are numerous and complex in composition. In the past, more than 150 compounds have been isolated from it, mainly including polysaccharides, triterpenoids, sterols, alkaloids, furan derivatives, amino polypeptides, and inorganic elements. Different geographical locations (longitude and latitude), different seeds, different growth environments, as well as different temperature, humidity, and light intensity will have a significant impact on the content, proportion, and presence or absence of the medicinal ingredients mentioned in the present invention in Ganoderma lucidum. Summary of the Invention

[0003] The purpose of the present invention is to provide a polysaccharide compound with a clear molecular structure, having anti-tumor efficacy and capable of eliminating the toxic and side effects of chemotherapy.

[0004] The present invention aims to solve the treatment problem of advanced cancer patients that has not been overcome internationally yet. Advanced cancer patients refer to those who have lost the surgical conditions, have a survival period of only three to six months, and still have the conditions for chemotherapy. The treatment problem refers to enabling advanced cancer patients to restore their appetite at the fastest speed (generally two to three weeks), making the tumor mass shrink or stop growing, and when used in combination with chemotherapy drugs, the toxic and side effects produced by the chemotherapy drugs on the human body can be basically eliminated. When used in long-term combination with chemotherapy drugs, it can achieve the purpose of basically eliminating cancer cells or controlling the number of cancer cells within the safe range for high-quality survival of the human body.

[0005] Another important function of the medicinal ingredient referred to in the present invention is that it can prevent the mutation of human normal cells and the generation of cancer cells.

[0006] Another important function of the medicinal ingredient referred to in the present invention is that when used in combination with chemotherapy drugs, it shows extremely excellent treatment effects on lung cancer, liver cancer, and breast cancer, showing a certain broad spectrum.

[0007] The present invention is realized as follows. A method for extracting Ganoderma lucidum polysaccharide GLP-3, the method comprising the following steps:

[0008] S1. Remove dust from Ganoderma lucidum and dry it, then perform crushing treatment to make Ganoderma lucidum powder;

[0009] S2. Place the crushed Ganoderma lucidum in a closed container and mix it with water and heat, and fully blend the Ganoderma lucidum powder and water into a medicinal juice solution under high temperature and high pressure;

[0010] S3. Use membrane concentration technology to separate the medicinal juice solution to obtain a concentrated solution with medicinal components and incompletely fused medicinal residues;

[0011] S4. Mix the concentrated solution containing medicinal components with pure water to form an aqueous solution with a preset concentration, and after multiple column chromatography separations and concentration and lyophilization, obtain Ganoderma lucidum polysaccharide GLP-3 with medicinal components.

[0012] A further technical solution of the present invention is that in step S2, the Ganoderma lucidum powder is mixed with water and heated sufficiently to 105-200 °C, and the boiling time lasts for 2-6 h. In the closed container, the Ganoderma lucidum powder and water are fully fused into a mixed medicinal liquid under high temperature and high pressure environment.

[0013] A further technical solution of the present invention is that in step S2, the Ganoderma lucidum powder is mixed with water and heated sufficiently to 105-170 °C, and the boiling time lasts for 3-6 h. In the closed container, the Ganoderma lucidum powder and water are fully fused into a mixed medicinal liquid under high temperature and high pressure environment.

[0014] A further technical solution of the present invention is that in step S4, the concentrated liquid containing medicinal components is mixed with pure water to a concentration ratio of 1:2-1:5.

[0015] A further technical solution of the present invention is that in step S3, the aqueous solution containing medicinal components extracted is used with membrane concentration technology to remove the Ganoderma lucidum residues therein to obtain a concentrated liquid or paste of medicinal components.

[0016] A further technical solution of the present invention is that in step S1, the Ganoderma lucidum is rinsed with clean water to remove the floating dust on the surface, dried at a high temperature of 105 °C through a drying device, and the dried Ganoderma lucidum is placed in a crushing device for crushing to obtain Ganoderma lucidum powder, and the Ganoderma lucidum powder is larger than 60 mesh.

[0017] A further technical solution of the present invention is that in step S2, the mixed liquid in the closed container is heated to 105 °C, 110 °C, 115 °C, 120 °C, 125 °C, 130 °C, 135 °C, 140 °C, 145 °C, 150 °C, 155 °C, 160 °C, 165 °C or 170 °C, and the boiling time is 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h or 6 h. In the closed container, the Ganoderma lucidum powder and water are fully fused into a mixed medicinal liquid under high temperature and high pressure environment.

[0018] Another object of the present invention is to provide a Ganoderma lucidum polysaccharide GLP-3, and the structural formula of the Ganoderma lucidum polysaccharide GLP-3 Molecular formula (C 66 H 110 O 55 ) n , wherein, n = 30-46.

[0019] A further technical solution of the present invention is that n is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46.

[0020] Another object of the present invention is to provide an application of Ganoderma lucidum polysaccharide GLP-3. The Ganoderma lucidum polysaccharide GLP-3 has good water solubility, is easily absorbed by the human body, has anti-tumor drug 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 and side effects caused by chemotherapy drugs on the human body and has an effect on controlling, reducing tumor masses, and reducing and eliminating tumor cells.

[0021] The beneficial effects of the present invention are as follows: The extraction process of this method is simple, the polysaccharide extraction rate is high, the production cost is low, and the operation is simple; the Ganoderma lucidum polysaccharide has good solubility and is easily absorbed by the human body, so as to play its anti-tumor role. Description of the Drawings

[0022] Figure 1 is a flowchart of the method for extracting Ganoderma lucidum polysaccharide GLP-3 provided by the embodiment of the present invention.

[0023] Figure 2 is a schematic diagram of the lgMp-RT (peak molecular weight) calibration curve provided by the embodiment of the present invention.

[0024] Figure 3 is a schematic diagram of the lgMp-RT (weight average molecular weight) calibration curve provided by the embodiment of the present invention.

[0025] Figure 4 is a schematic diagram of the lgMp-RT (number average molecular weight) calibration curve provided by the embodiment of the present invention.

[0026] Figure 5 is a schematic diagram of the molecular weight spectrum of Ganoderma lucidum polysaccharide GLP-3 provided by the embodiment of the present invention.

[0027] Figure 6 is a schematic diagram of the infrared spectrum of polysaccharide provided by the embodiment of the present invention.

[0028] Figure 7 is a schematic diagram of the ion chromatogram of the 16-sugar mixed standard provided by the embodiment of the present invention Figure 1 .

[0029] Figure 8 is a schematic diagram of the ion chromatogram of the 16-sugar mixed standard provided by the embodiment of the present invention Figure 2 .

[0030] Figure 9 is the GCMS chromatogram of the sample (PMAA) provided by the embodiment of the present invention.

[0031] Figure 10 Schematic diagram of the result analysis of polysaccharide methylated sugar alcohol acetate (PMAA) provided by an embodiment of the present invention Figure 1 .

[0032] Figure 11 Schematic diagram of the result analysis of polysaccharide methylated sugar alcohol acetate (PMAA) provided by an embodiment of the present invention Figure 2 .

[0033] Figure 12 Schematic diagram of the result analysis of polysaccharide methylated sugar alcohol acetate (PMAA) provided by an embodiment of the present invention Figure 3 .

[0034] Figure 13 Schematic diagram of the result analysis of polysaccharide methylated sugar alcohol acetate (PMAA) provided by an embodiment of the present invention Figure 4 .

[0035] Figure 14 Schematic diagram of the result analysis of polysaccharide methylated sugar alcohol acetate (PMAA) provided by an embodiment of the present invention Figure 5 .

[0036] Figure 15 Schematic diagram of the result analysis of polysaccharide methylated sugar alcohol acetate (PMAA) provided by an embodiment of the present invention Figure 6 .

[0037] Figure 16 1H NMR spectrum schematic diagram provided by an embodiment of the present invention

[0038] Figure 17 13C NMR spectrum schematic diagram provided by an embodiment of the present invention

[0039] Figure 18 Dept135 spectrum schematic diagram provided by an embodiment of the present invention

[0040] Figure 19 HH-COSY schematic diagram provided by an embodiment of the present invention

[0041] Figure 20 HSQC spectrum provided by an embodiment of the present invention

[0042] Figure 21 HMBC spectrum provided by an embodiment of the present invention

[0043] Figure 22 NOESY schematic diagram provided by an embodiment of the present invention

[0044] Figure 23 Schematic diagram of the control group of the GLP-3 combined with chemotherapy on LLC lung cancer-bearing mouse model provided by an embodiment of the present invention

[0045] Figure 24It is a schematic diagram of the cisplatin 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.

[0046] Figure 25 It 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.

[0047] Figure 26 It 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.

[0048] Figure 27 It is a schematic diagram of the tumor model control 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 28 It is a schematic diagram of the tumor cisplatin 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.

[0050] Figure 29 It is a schematic diagram of the tumor 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.

[0051] Figure 30 It is a schematic diagram of the tumor 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.

[0052] Figure 31 It is a schematic diagram of the tumor model control 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.

[0053] Figure 32 It is a schematic diagram 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.

[0054] Figure 33 It 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.

[0055] Figure 34 It is a schematic diagram of the cisplatin + GLP-3 high-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 35 It is a schematic diagram of the appearance of the tumor model control 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.

[0057] Figure 36 It is a schematic diagram of the appearance of the cisplatin group of tumors in H22 hepatoma-bearing mice treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided by an embodiment of the present invention.

[0058] Figure 37 It is a schematic diagram of the appearance of the cisplatin + GLP-3 low-dose group of tumors in H22 hepatoma-bearing mice treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided by an embodiment of the present invention.

[0059] Figure 38 It is a schematic diagram of the appearance of the cisplatin + GLP-3 high-dose group of tumors in H22 hepatoma-bearing mice treated with Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy provided by an embodiment of the present invention.

[0060] Figure 39 It is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on tumors in H22 orthotopic liver cancer mice provided by an embodiment of the present invention (binning: 8×8; T = 30s).

[0061] Figure 40 It is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on tumors in H22 orthotopic liver cancer mice provided by an embodiment of the present invention.

[0062] Figure 41 It is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on the liver of H22 orthotopic liver cancer mice provided by an embodiment of the present invention (×200).

[0063] Figure 42 It is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on the spleen of H22 orthotopic liver cancer mice provided by an embodiment of the present invention (×200).

[0064] Figure 43 It is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on the thymus of H22 orthotopic liver cancer mice provided by an embodiment of the present invention (×200). Detailed implementation manners

[0065] The embodiments of the present invention are described in detail below. The examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions from beginning to end. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention, but should not be construed as limiting the present invention.

[0066] As Figure 1 shown, the flowchart of the method for extracting Ganoderma lucidum polysaccharide GLP-3 provided by the present invention is described in detail as follows:

[0067] Step S1: Wash the picked Ganoderma lucidum or the Ganoderma lucidum that has been preliminarily and simply processed with clean water on a cleaning device to remove the floating dust on its surface. After removing the floating dust, transfer the Ganoderma lucidum to a drying device for high-temperature drying at a drying temperature of 105°C. Place the high-temperature dried Ganoderma lucidum on a crusher or grinder to crush it into Ganoderma lucidum powder. Screen the crushed Ganoderma lucidum powder. Select the Ganoderma lucidum powder particles larger than 60 mesh, and return the Ganoderma lucidum powder particles smaller than 60 mesh to the crusher or grinder for re-crushing. Repeat this process multiple times until the crushed Ganoderma lucidum powder particles meet the specified requirements.

[0068] Step S2: Mix the qualified Ganoderma lucidum powder with pure water and place it in a sealed container. Heat the sealed container. As the continuous heating temperature of the sealed container rises, a high-temperature and high-pressure environment is formed inside the sealed container, making it easier to fully blend the Ganoderma lucidum powder and water to form a mixed liquid. When heating the sealed container to a high temperature between 105°C and 200°C, the boiling time is 2 - 6 hours. Preferably, it is heated to a high temperature between 105°C and 170°C, and the boiling time is 3 - 6 hours. More preferably, it is heated to 105°C, 110°C, 115°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, 170°C, 175°C, 180°C, 185°C, 190°C, 195°C or 200°C, and the boiling time is 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours or 6 hours, so that the mixed liquid is fully blended. The sealed container is a reaction kettle.

[0069] Step S3: Centrifuge the aqueous solution containing the medicinal ingredients to remove the residue, and concentrate the medicinal liquid using membrane concentration technology to obtain a concentrated liquid.

[0070] Step S4: Prepare the concentrated liquid containing the medicinal ingredients to a certain concentration, separate it by column chromatography, and concentrate and freeze-dry it to obtain the medicinal ingredient Ganoderma lucidum polysaccharide GLP-3.

[0071] This extraction method has a simple process, a high polysaccharide extraction rate, low production costs, and is easy to operate.

[0072] Another object of the present invention is to provide a Ganoderma lucidum polysaccharide GLP-3, and the structural formula of the Ganoderma lucidum polysaccharide GLP-3 Molecular formula C 66 H 110 O 55 ) n , where n = 30 - 46.

[0073] n is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46.

[0074] After obtaining Ganoderma lucidum polysaccharide GLP-3 with the above structure, determination experiments were carried out on it, and the following report results were obtained.

[0075] II. Molecular Weight Determination

[0076] 1. Experimental Purpose

[0077] Determine the molecular weight and purity of polysaccharides by HPGPC.

[0078] 2. Experimental Materials

[0079] 2.1 Instruments

[0080]

[0081] 2.2 Materials

[0082] Reagent Manufacturer Batch Number Item Number Grade Expiry Date NaCl ACROS A0356762 139725000 ACROS In 2022

[0083] 2.3 Standards

[0084]

[0085]

[0086] 3. Experimental Procedures

[0087] 3.1 Reagent Preparation

[0088] Reagent Name Preparation Method Storage Conditions Shelf Life 0.05M NaCl Solution Precisely prepared, filtered through a 0.45μm filter membrane, and degassed by ultrasonic for 10 min RT One month

[0089] 3.2 Sample and Standard Solution Preparation

[0090] Precisely weigh the sample and the standard. The sample is prepared into a 5 mg / ml solution, centrifuged at 12000 rpm for 10 min, and the supernatant is filtered through a 0.22 μm microporous filter membrane. Then the sample is transferred to a 1.8 ml injection vial.

[0091] 3.3 Chromatographic Method

[0092] Chromatographic column: BRT105-104-102 tandem gel column (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.

[0093] 4. Experimental Results

[0094] As Figure 2 - 4 shown, the calibration curves of lgMp-RT (peak molecular weight), lgMw-RT (weight-average molecular weight), and lgMn-RT (number-average molecular weight) were obtained.

[0095] The calibration curve equation of lgMp-RT is: y = -0.184x + 11.752R 2 = 0.9956;

[0096] The calibration curve equation of lgMw-RT is: y = -0.1961x + 12.315R 2 = 0.9934;

[0097] The calibration curve equation of lgMn-RT is: y = -0.1818x + 11.589R 2 = 0.992;

[0098] According to the standard curve, the calculation formula is obtained and then the molecular weight of each sample is calculated. The molecular weight spectrum of the sample is as Figure 5 shown, and the calculation results are shown in the following table.

[0099] SampleID RT (min) lgMp lgMw lgMn Mp Mw Mn Peak Area Ratio % 37.747 4.8 4.9 4.7 64055 81811 53284 100

[0100] Among them, 46.2 min is the peak of the mobile phase.

[0101] II. Monosaccharide composition determination experiment

[0102] 1. Experimental purpose

[0103] Use an ion chromatograph to determine the monosaccharide composition.

[0104] 2. Experimental principle

[0105] Based on the electrochemically active properties of carbohydrate molecules and their ionization in strong alkaline solutions. Carbohydrate compounds are weak acids with pKa > 11. In the eluent with high pH value, they will exist partially or entirely in the form of anions. According to the differences in ion exchange caused by the differences in pKa of different carbohydrate compounds and the differences in hydrophobic interactions between certain carbohydrates and anion exchange resins, high-performance anion exchange separation of carbohydrate compounds is achieved, and then the current generated by the oxidation reaction of the hydroxyl groups in the sugar molecular structure on the surface of the gold electrode is detected.

[0106] 3. Experimental materials

[0107] 3.1 Instruments

[0108] Instrument Name Manufacturer Model Ion Chromatograph ThermoFisher ICS5000 Electric Thermostatic Blast Drying Oven Lichen Technology 101 - 1BS Nitrogen Blower Lichen Technology UGC - 24M Electronic Analytical Balance Sartorius BS210S Centrifuge ThermoFisher D-37520 Pipette DRAGONLAB 19050983

[0109] 3.2 Reagents

[0110] Reagent Manufacturer Batch Number Item Number Grade Trifluoroacetic Acid ACROS A0356762 139725000 AR 50% Sodium Hydroxide Solution Alfa Aesar Z21E036 33382 GR Sodium Acetate ThermoFishe 191126 059326 GR

[0111] 3.3 Standards

[0112]

[0113]

[0114] 4. Experimental methods

[0115] 4.1 Reagent preparation

[0116] Reagent Name Preparation Method Storage Conditions 15mM NaOH Solution 2.4g of 50% Sodium Hydroxide Solution, 2L of water RT 15mM NaOH & 100mM NaOAC Solution 1.2g of 50% Sodium Hydroxide Solution, 8.2g of NaOAC, 1L of water RT

[0117] 4.2 Preparation and calculation method of standard solutions

[0118] Take 16 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, D-galactosamine hydrochloride, D-glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, mannuronic acid) to prepare a standard stock solution.

[0119] Precisely prepare concentration standards of each monosaccharide standard solution as a mixed standard. According to the absolute quantification method, determine the mass of different monosaccharides, and calculate the molar ratio based on the molar mass of the monosaccharides.

[0120] 4.3 Sample preparation

[0121] Precisely weigh 5 mg of the sample and place it in an ampoule. Add 2 ml of 3M TFA and hydrolyze at 120 °C for 3 h. Accurately pipette the acid hydrolysis solution into a tube and blow it dry under nitrogen. Add 5 ml of water and vortex to mix evenly. Pipette 50 μL and add 950 μL of deionized water, then centrifuge at 12000 rpm for 5 min. Take the supernatant for IC analysis.

[0122] 4.4 Chromatographic method

[0123] Chromatographic column: Dionex Carbopac TM PA20 (3 * 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 μL; Column temperature: 30 °C; Detector: Electrochemical detector.

[0124] 4.5 Standard sequence

[0125] 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

[0126] C (standard) / A (standard) = C (sample) / A (sample)

[0127] 5. Experimental results

[0128] Mixed standard: Solvent peaks: The peak of sodium hydroxide is at 2.0 min, and the peak of sodium acetate is at 41 min. As Figure 6 、 7 shown.

[0129] Name RT Molar Ratio Fucose 5.659 0.000 Galactosamine Hydrochloride 10.084 0.000 Rhamnose 10.475 0.000 Arabinose 11.092 0.000 Glucosamine Hydrochloride 12.367 0.000 Galactose 13.767 0.000 Glucose 15.475 1.000 N - Acetyl - D - Glucosamine 16.792 0.000 Xylose 17.834 0.000 Mannose 18.117 0.000 Fructose 20.534 0.000 Ribose 22.484 0.000 Galacturonic Acid 45.125 0.000 Guluronic Acid 45.95 0.000 Glucuronic Acid 48.509 0.000 Mannuronic Acid 50.992 0.000

[0130] III. Experiment for Determining the Linkage Mode of Polysaccharides

[0131] 1. Experiment Purpose

[0132] Determine the linkage mode of the polysaccharide sample by GC-MS after methylation and other derivatizations.

[0133] 2. Experiment Materials

[0134] 2.1 Instruments

[0135] Instrument Name Manufacturer Model Rotary Evaporator Zhengzhou Great Wall Scientific & Industrial Co., Ltd R - 1001VN Nitrogen Blower Lichen Technology UGC - 24M Magnetic Stirrer DLAB MS7 - H550 - Pro Vacuum Drying Oven Lichen Technology 101 - 1BS Gas Chromatograph - Mass Spectrometer Agilent 6890-5973

[0136] 2.2 Reagents

[0137] Reagent Manufacturer Batch Number Item Number Grade Trifluoroacetic Acid ACROS A0356762 139725000 AR Methyl Iodide Adamas P1345479 01111630 AR Sodium Borohydride Aldrich MKCD7945 205591 AR Ethyl Acetate Wokai 08050003 40065982 AR Acetic Anhydride Hushi 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

[0138] 3. Experiment Methods

[0139] 3.1 Reagent Preparation

[0140] Reagent Name Preparation Method Storage Conditions 3M Trifluoroacetic Acid 1V Trifluoroacetic Acid + 3V Water Store in a 5°C Refrigerator Sodium Hydride Dry Powder Wash with 60% Sodium Hydride in Hexane Store at Room Temperature, Dry Sodium Borodeuteride Sodium Hydroxide Solution 20mg + 20mM NaOH Solution Store Sealed 20% Acetic Acid in Methanol Solution 1V Glacial Acetic Acid + 4V Water Store in a 5°C Refrigerator Polysaccharide Methylation Kit Solution A, Anhydrous Base Solution Store in a 5°C Refrigerator Solution B, Iodomethane Solution

[0141] 3.2 Sample Methylation

[0142] After methylation, hydrolysis, and acetylation of the sample, it is determined by GC-MS and compared with the standard mass spectrometry library.

[0143] Weigh the polysaccharide sample (2 - 3 mg) and place it in a glass reaction flask. Add 1 mL of anhydrous DMSO, quickly add Solution A of the methylation reagent, seal it, dissolve it under ultrasonic action, and then add Solution B of the methylation reagent. React in a magnetic stirring water bath at 30 °C for 60 min. Finally, add 2 mL of ultrapure water to the above mixture to terminate the methylation reaction.

[0144] Take the methylated polysaccharide, add 1 mL of 2 M trifluoroacetic acid (TFA) and hydrolyze for 90 min. Evaporate to dryness using a rotary evaporator. Add 2 mL of double-distilled water to the residue, reduce it with 60 mg of sodium borohydride for 8 hours, neutralize it with glacial acetic acid, rotary evaporate, dry it in an oven at 101 °C, then add 1 mL of acetic anhydride and acetylate at 100 °C for 1 h, and cool it. Then add 3 mL of toluene, concentrate it under reduced pressure and evaporate to dryness, repeating 4 - 5 times to remove the excess acetic anhydride.

[0145] Dissolve the acetylated product in 3 mL of CH2Cl2 and transfer it to a separatory funnel. Add a small amount of distilled water, shake it well, and remove the upper aqueous solution. Repeat this 4 times. Dry the CH2Cl2 layer with an appropriate amount of anhydrous sodium sulfate, make the volume up to 10 mL, and put it into a liquid phase vial. Analyze the acetylated product sample using a Shimadzu GCMS-QP 2010 gas chromatography - mass spectrometry instrument;

[0146] GC-MS conditions: RXI-5 SIL MS chromatographic column, 30 m * 0.25 mm * 0.25 μm; The temperature programming conditions are as follows: The initial temperature is 120 °C, and it is heated to 250 °C at a rate of 3 °C / min; Hold for 5 min; The inlet temperature is 250 °C, the detector temperature is 250 °C, the carrier gas is helium, and the flow rate is 1 mL / min.

[0147] 4. Experimental results

[0148] The GCMS chromatogram of the sample (PMAA) is as Figure 9 shown.

[0149] The result analysis of polysaccharide methylated alditol acetate (PMAA) is shown in the following table and as Figures 10 - 15 shown.

[0150]

[0151] IV. NMR Spectra Analysis and Interpretation

[0152] 1. Experimental Materials and Instruments

[0153] Heavy water (D2O, 99.9%) and deuterated acetone are used as internal references; Freeze dryer, nuclear magnetic resonance spectrometer Bruker 600M (Nuclear Magnetic Resonance, NMR);

[0154] 2. Experimental Procedures

[0155] Weigh 50 mg of the polysaccharide sample, dissolve it in 0.5 ml of heavy water and freeze-dry it. Then dissolve the lyophilized powder in 0.5 ml of heavy water again and continue to freeze-dry it. Repeat the above process to fully exchange the labile hydrogens. Then dissolve the sample in 0.5 ml of heavy water and measure the 1 1H NMR spectrum, 13 13C NMR spectrum, DEPT135 one-dimensional spectrum and two-dimensional spectra at room temperature of 25 °C using a 600 MHz nuclear magnetic resonance spectrometer.

[0156] 3. Experimental Results

[0157] The hydrogen spectrum signals are mainly concentrated between 3.0 and 5.5 ppm. The signals of δ 3.2 - 4.0 ppm are the sugar ring proton signals, and the main anomeric proton peaks δ 4.42, 4.45, 4.49, 4.50, 4.66, 4.71 have signal peaks concentrated in the region of 4.3 - 5.5 ppm. As Figure 16 shown.

[0158] The carbon spectrum analysis is at 1313C NMR (201 MHz, D2O): The 13C NMR signals are mainly concentrated between 60 - 120 ppm. By observing the carbon spectrum, the main anomeric carbon signal peaks are δ103.82, 103.89, 104.02, 104.02, 104.12, 104.21. The anomeric carbon region is mainly between δ93 - 105. And the signals at δ72.72, 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, 68.79 ppm region. According to the results of monosaccharide composition, this polysaccharide is composed of glucose. It shows that this polysaccharide is mainly glucan. As Figure 17 shown.

[0159] Analysis of the Dept135 spectrum shows that the peaks at 60.70, 68.82, 61.66, 62.04, 70.28, 68.79 ppm are inverted peaks, indicating the chemical shifts of C6. As Figure 18 shown.

[0160] As Figures 19 - 22 shown, through the HSQC spectrum, the anomeric carbon signal can be observed as δ103.94, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ4.71. Through HH - COSY, the signal of H1 - 2 is 4.71 / 3.44; the signal of H2 - 3 is 3.44 / 3.66; the signal of H3 - 4 is 3.66 / 3.40. We can infer that H1, H2, H3, H4 are δ4.71, 3.44, 3.66, 3.40 respectively. The corresponding δ104.04, 74.55, 85.9, 76.63. The corresponding C5 is 77.41; the chemical shift of C6 is δ62.04. Therefore, this signal should be assigned to the glycosidic bond →3)-β - Glcp-(1→.

[0161] From the HSQC spectrum, the anomeric carbon signal was observed at δ104.21, and the corresponding anomeric hydrogen signal in the HSQC spectrum was δ4.42. From the HH-COSY, the signals of H1-2 were 4.42 / 3.23; the signals of H2-3 were 3.23 / 3.39; the signals of H3-4 were 3.39 / 3.55. We can infer that H1, H2, H3, and H4 were δ4.42, 3.23, 3.39, and 3.55 respectively, and their corresponding C1-4 were δ104.21, 74.52, 76.74, and 76.41. And from the NOESY spectrum, relevant peaks were observed between δ4.43 and 3.39, 3.55, 3.75, 4.11. Combining Dept135 with HSQC, it can be determined that the peaks at δ3.75 and 4.11 belong to H6a,b, and H5 was 3.31 ppm. The corresponding C5 was 70.89; the chemical shift of C6 was δ70.19, and the corresponding H6a was δ3.75, 4.11. Therefore, this signal should be attributed to the glycosidic bond →6)-β-Glcp-(1→.

[0162] According to similar rules and combining HMBC and NOESY, the signals of all glycosidic bonds were attributed as shown in the following table:

[0163] Attribution of hydrogen and carbon signals

[0164]

[0165] Main chain analysis:

[0166] In the HMBC spectrum, based on the one-dimensional and two-dimensional NMR spectra, we attributed the signals of the glycosidic bonds of the polysaccharide; there was a relevant signal peak between the anomeric hydrogen of the glycosidic bond →6)-β-D-Glcp-(1→ and its own C6; indicating the existence of the linkage mode of →6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→.

[0167] There was a relevant signal peak between the anomeric hydrogen of the glycosidic bond →6)-β-D-Glcp-(1→ and the C6 of →3,6)-β-D-Glcp-(1→; indicating the existence of the linkage mode of →6)-β-D-Glcp-(1→3,6)-β-D-Glcp-(1→.

[0168] There was a relevant signal peak between the anomeric hydrogen of the glycosidic bond →3,6)-β-D-Glcp-(1→ and the C6 of →4,6)-β-D-Glcp-(1→; indicating the existence of the linkage mode of →3,6)-β-D-Glcp-(1→4,6)-β-D-Glcp-(1→.

[0169] Branched chain analysis:

[0170] The anomeric carbon of the glycosidic bond →3)-β-D-Glcp-(1→ has a correlated signal peak with the H3 of →3,6)-β-D-Glcp-(1→; indicating the existence of the linkage mode of →3)-β-D-Glcp-(1→3,6)-β-D-Glcp-(1→.

[0171] In the NOESY spectrum,

[0172] The anomeric hydrogen of the glycosidic bond β-D-Glcp-(1→ has a correlated signal peak with the H4 of →4)-β-D-Glcp-(1→; indicating the existence of the linkage mode of β-D-Glcp-(1→4)-β-D-Glcp-(1→.

[0173] The anomeric hydrogen of the glycosidic bond β-D-Glcp-(1→ has a correlated signal peak with the H3 of →3)-β-D-Glcp-(1→; indicating the existence of the linkage mode of β-D-Glcp-(1→3)-β-D-Glcp-(1→.

[0174] The anomeric hydrogen of the glycosidic bond →4)-β-D-Glcp-(1→ has a correlated signal peak with the H4 of →4,6)-β-D-Glcp-(1→; indicating the existence of the linkage mode of →4)-β-D-Glcp-(1→4,6)-β-D-Glcp-(1→.

[0175] 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 respectively connected to the main chain through the o-3 of →3,6)-β-D-Glcp-(1→ and the o-4 of →4,6)-β-D-Glcp-(1→, and its molecular structural formula is shown as follows.

[0176]

[0177] Another object of the present invention is to provide an application of Ganoderma lucidum polysaccharide GLP-3. The Ganoderma lucidum polysaccharide GLP-3 has good water solubility, is easily absorbed by the human body, has anti-tumor efficacy and has an effect on preventing the occurrence of tumors in humans. Especially when used in combination with chemotherapy drugs, it can eliminate the toxic and side effects caused by chemotherapy drugs to the human body and has an effect on the control, reduction of tumor masses and the reduction and elimination of tumor cells.

[0178] The Ganoderma lucidum polysaccharide GLP-3 is applied to drugs for inhibiting tumor spread or drugs for enhancing human immunity.

[0179] Experiment on the anti-tumor effect and data of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on LLC lung cancer-bearing mice

[0180] Experimental purpose

[0181] C57 mice were subcutaneously inoculated with homogenates of LLC lung cancer tumor blocks under the right axilla to prepare tumor-bearing mice, and the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-3 on lung cancer tumor-bearing mice was studied to provide experimental basis for its clinical research.

[0182] Experimental materials

[0183] Test samples

[0184] Ganoderma lucidum polysaccharide GLP-3 was provided by Shenzhen Aolimei Tumor Medical Technology Co., Ltd.

[0185] Positive control

[0186] Cisplatin, batch number: E2128081, product of Shanghai Aladdin Biochemical Technology Co., Ltd.

[0187] Experimental animals

[0188] 75 SPF-grade male C57 mice, weighing 14 - 16 g, were provided by Guangdong Medical Experimental Animal Center. The production license number of experimental animals is: SCXK(Guangdong)2022 - 0002, and the quality certificate number of experimental animals is: 44007200103827.

[0189] Main reagents

[0190] PBS buffer was prepared by Shenzhen Aolimei Tumor Medical Technology Co., Ltd.; fetal bovine serum, product of Zhejiang Tianhang Biotechnology Co., Ltd.; DMEM medium, product of Gibco; 0.25% trypsin, product of Gibco.

[0191] Main instruments

[0192] Vernier calipers, product of Shanghai Tool Works Co., Ltd.; I-2000 type balance, Dongguan Nancheng Changxie Electronic Products Factory; ophthalmic scissors and forceps, product of Shanghai Jinzhong Medical Instrument Co., Ltd.; CCL-170B-8 carbon dioxide incubator, product of ESCO in Singapore; Luna-Ⅱ cell counter, product of Nanjing Hengqiao Instrument Co., Ltd., JRA-35S handheld homogenizer, product of Wuxi Jieran Instrument Equipment Co., Ltd.

[0193] Experimental methods

[0194] Take normally growing LLC cells and inoculate them subcutaneously on the left shoulder and back of 5 healthy male C57 mice. When the tumor grows to a volume of 2000 - 3000 mm 3 3, dissect the tumor mass and make it into a homogenate suspension, and inject it subcutaneously under the axilla of 60 healthy male C57 mice to make a solid tumor model. When the average tumor volume of all mice is 150 mm 3Around [specific time], the mice were randomly grouped according to the tumor volume, and then given the corresponding drugs or drug solvents by gavage or intraperitoneal injection for 19 consecutive days. The long and short diameters of the tumors were measured every 3 days to calculate the tumor volume, and the body weights of the mice were weighed every 3 days. At the end of the experiment, the tumors, spleens, and thymuses of the mice were dissected and weighed, and the tumor index, spleen index, and thymus index were calculated.

[0195] Dose Design

[0196] Based on the results of previous experiments, the Ganoderma lucidum polysaccharide GLP-3 was designed. The low dose was 50 mg / kg, and the high dose was 150 mg / kg. The corresponding drugs were given according to Table 1.

[0197] Dose design basis for cisplatin: According to the clinical dosage of cisplatin, which should not exceed 100 mg / m per person per day 2 and the tolerance of mice to cisplatin, a dose of 4 mg / kg was selected as the administration dose.

[0198] Table 1 Experimental grouping and dose design

[0199]

[0200] Detection Index

[0201] Efficacy Index

[0202] Relative Tumor Inhibition Rate

[0203] Relative Tumor Inhibition Rate (%) = (1 - T RTV / C RTV ) × 100%. Where T RTV is the relative tumor volume of the experimental group, and C RTV is the relative tumor volume of the model control group. Relative Tumor Volume RTV = V t / V0, V t is the tumor volume of the mice on the t-th day of drug administration, and V0 is the tumor volume of the mice at the time of grouping. Evaluation criteria: Relative Tumor Inhibition Rate ≥ 40%, and P < 0.05 after statistical analysis is the effective inhibition level.

[0204] Tumor Growth Inhibition Rate

[0205] 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 P < 0.05 in statistical analysis is the effective inhibition level.

[0206] Spleen and thymus organ coefficients: After the last drug administration, the spleens, thymuses, and tumors were weighed to calculate the organ coefficients.

[0207] Tumor Index (%) = tumor weight / body weight × 100%.

[0208] Immune organ index (mg / g) = organ mass / body weight × 1000.

[0209] Data processing and statistical analysis

[0210] SPSS 17.0 was used for statistical analysis. The level of statistical significance was set at P ≤ 0.05. Measurement data were expressed as mean ± standard deviation and the Leven’s test method 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), the 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 during evaluation

[0211] Experimental results

[0212] Animal death situation

[0213] As shown in Table 2, the mortality rate of mice in each group of this experiment was 0.

[0214] Table 2 Statistics of the number of surviving animals and mortality rate in each group

[0215]

[0216] Effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the body weight of LLC lung cancer-bearing mice

[0217] 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, D9 - D19. The body weight of mice in the cisplatin group and the high-dose cisplatin + GLP-3 group was significantly decreased on D0, D6 - D19. The body weight of mice in the low-dose cisplatin + GLP-3 group was significantly decreased on D6 - D19.

[0218] 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 decreased on D6 - D19. The body weight of mice in the low-dose cisplatin + GLP-3 group was significantly decreased on D3 - D19.

[0219] Compared with the cisplatin group, the body weight of mice in the low-dose cisplatin + GLP-3 group was significantly decreased on D12 - D19.

[0220] Table 3 Effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the body weight of LLC lung cancer-bearing mice

[0221]

[0222] 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.

[0223] Effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on tumor volume in LLC lung cancer-bearing mice

[0224] As shown in Table 4, compared with the model control group, the tumor volume in the cisplatin group significantly decreased from D9 to D19, and the tumor volume in the low-dose cisplatin + GLP-3 group and the high-dose cisplatin + GLP-3 group significantly decreased from D6 to D19.

[0225] Compared with the cisplatin group, the tumor volume in the low-dose cisplatin + GLP-3 group significantly decreased from D9 to D19, and the tumor volume in the high-dose cisplatin + GLP-3 group significantly decreased from D12 to D19.

[0226] Table 4 Effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on tumor volume in LLC lung cancer-bearing mice

[0227]

[0228] Note: Compared with the model control group, + P < 0.05; compared with the cisplatin group, # P < 0.05.

[0229] Effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on relative tumor inhibition rate in LLC lung cancer-bearing mice

[0230] As shown in Table 5 and Table 6, compared with the model control group, the relative tumor inhibition rate in the cisplatin group was above 40% from D9 to D19, which were 54.6%, 63.8%, 77.3%, and 79.5% respectively; the relative tumor inhibition rate in the low-dose cisplatin + GLP-3 group was above 50% from D6 to D19, which were 51.9%, 70.0%, 80.1%, 87.4%, and 92.2% respectively; the relative tumor inhibition rate in the high-dose cisplatin + GLP-3 group was above 40% from D6 to D19, which were 43.3%, 67.1%, 74.9%, 82.3%, and 88.0% respectively.

[0231] Compared with the cisplatin group, the relative tumor inhibition rate in the low-dose cisplatin + GLP-3 group was 44.9%, 44.6%, and 62.3% from D12 to D19 (P < 0.05), and the relative tumor inhibition rate in the high-dose cisplatin + GLP-3 group was 30.6%, 22.3%, and 41.6% from D12 to D19 (P < 0.05).

[0232] Table 5 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the relative tumor inhibition rate (compared with the model control group) of LLC lung cancer-bearing mice

[0233]

[0234]

[0235] Note: Compared with the model control group, + P < 0.05.

[0236] Table 6 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the relative tumor inhibition rate (compared with the cisplatin group) of LLC lung cancer-bearing mice

[0237]

[0238] Note: Compared with the cisplatin group, # P < 0.05.

[0239] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the organ index and tumor growth inhibition rate of LLC lung cancer-bearing mice

[0240] As shown in Table 7, compared with the model group, the tumor index, spleen index, and thymus index in the cisplatin group, cisplatin + GLP-3 low-dose group, and cisplatin + GLP-3 high-dose group were all significantly decreased. Compared with the cisplatin group, the tumor index in the cisplatin + GLP-3 low-dose group was significantly decreased. Compared with the normal group, the spleen index in the model control group was significantly increased, while that in the cisplatin + GLP-3 low-dose group was significantly decreased; the thymus index in the model control group, cisplatin group, cisplatin + GLP-3 low-dose group, and cisplatin + GLP-3 high-dose group was significantly decreased.

[0241] Compared with the model control group, the tumor growth inhibition rate in the cisplatin group was 74.4%, and those in the cisplatin + GLP-3 low-dose group and high-dose group were 91.7% and 84.0% respectively. Compared with the cisplatin group, the tumor growth inhibition rates in the cisplatin + GLP-3 low-dose group and high-dose group were 67.4% and 37.4% respectively.

[0242] Table 7 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the organ index and tumor growth inhibition rate of LLC lung cancer-bearing mice

[0243]

[0244] 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.

[0245] Such asFigure 23 , 24 , as shown in Figures 25 and 26, are pictures of tumor-bearing mice, and the corresponding groups are as follows: 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

[0246] As Figure 27 , 28 , as shown in Figures 29 and 30, are pictures of tumors of tumor-bearing mice, and the corresponding groups are as follows: Figure 27 : Model control group, Figure 28 : Cisplatin group, Figure 29 : Cisplatin + GLP-3 low-dose group, Figure 30 : Cisplatin + GLP-3 high-dose group.

[0247] Conclusion

[0248] 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.

[0249] Experiment on the anti-tumor effect and data of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on H22 hepatocarcinoma-bearing mice

[0250] Experimental purpose

[0251] Tumor-bearing mice were prepared by inoculating the homogenate of H22 hepatocarcinoma tumor mass under the right axilla of C57 mice to study the effect of Ganoderma lucidum polysaccharide GLP-3 on hepatocarcinoma-bearing mice and provide experimental basis for its clinical research.

[0252] Experimental materials

[0253] Test articles

[0254] Ganoderma lucidum polysaccharide GLP-3 was provided by Shenzhen Aolimei Tumor Medical Technology Co., Ltd.

[0255] Positive control

[0256] Cisplatin, batch number: E2128081, product of Shanghai Aladdin Biochemical Technology Co., Ltd.

[0257] Experimental animals

[0258] 65 SPF-grade male C57 mice, weighing 16 - 18 g, were provided by Guangdong Medical Experimental Animal Center. The production license number of experimental animals: SCXK(Yue)2022 - 0002, and the quality certificate number of experimental animals: 44007200103599.

[0259] Main reagents

[0260] PBS buffer solution, prepared by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.; fetal bovine serum, product of Zhejiang Tianhang Biotechnology Co., Ltd.; 1640 medium, product of Gibco; 0.25% trypsin, product of Gibco.

[0261] Main instruments

[0262] Vernier caliper, product of Shanghai Tool Works Co., Ltd.; I-2000 type balance, Dongguan Nancheng Changxie Electronic Products Factory; ophthalmic scissors and forceps, product of Shanghai Jinzhong Medical Instruments Co., Ltd.; CCL-170B-8 carbon dioxide incubator, product of ESCO, Singapore; Luna-II cell counter, product of Nanjing Hengqiao Instruments Co., Ltd., JRA-35S handheld homogenizer, product of Wuxi Jieruian Instrument Equipment Co., Ltd.

[0263] Experimental methods

[0264] Homogenate suspension of H22 liver cancer solid tumor was injected subcutaneously under the armpit of 50 healthy male C57 mice to make a solid tumor model. When the average tumor volume of all mice was about 200 mm 3 or so, they were randomly grouped according to the tumor volume, and given the corresponding drugs or drug solvents by gavage or intraperitoneal injection respectively, and the drugs were administered continuously for 22 days. The long and short diameters of the tumor were measured once every 3 days, the tumor volume was calculated, and the body weight of the mice was weighed once every 3 days. At the end of the experiment, the tumors, spleens and thymuses of the mice were dissected and weighed, and the tumor index, spleen index and thymus index were calculated.

[0265] Dose design

[0266] According to the previous experimental results, Ganoderma lucidum polysaccharide GLP-3 was designed, with the low dose being: 50 mg / kg and the high dose being: 150 mg / kg, and the corresponding drugs were given as shown in Table 8.

[0267] Dose design basis for cisplatin: According to the clinical dosage of cisplatin not exceeding 100 mg / m per person per day 2 and the tolerance of mice to cisplatin, a dose of 3 mg / kg was selected as the administration dose.

[0268] Table 8 Experimental grouping and dose design

[0269]

[0270] Detection indexes

[0271] Efficacy indexes

[0272] Relative tumor inhibition rate

[0273] Relative tumor inhibition rate (%) = (1 - T RTV / C RTV ) × 100%. Among them, TRTV is the relative tumor volume of the experimental group, C RTV is the relative tumor volume of the model control group. Relative tumor volume RTV = V t / V0, where V t is the tumor volume of the mice on the t-th day after drug administration, and V0 is the tumor volume of the mice at the time of grouping. Evaluation criteria: relative tumor inhibition rate ≥ 40%, and P < 0.05 by statistical analysis is the effective inhibition level.

[0274] Tumor growth inhibition rate

[0275] Tumor growth inhibition rate (%) = (1 - T / C) × 100%. Among them, 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 is the effective inhibition level.

[0276] Spleen and thymus organ coefficients: After the last drug administration, the spleen, thymus, and tumors were weighed and the organ coefficients were calculated.

[0277] Tumor index (%) = tumor weight / body weight × 100%.

[0278] Immune organ index (mg / g) = organ mass / body weight × 1000.

[0279] Data processing and statistical analysis

[0280] SPSS 17.0 was used for statistical analysis. The level of statistical significance was set at P ≤ 0.05. Measurement data were expressed as mean ± standard deviation and the Leven’s test method 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), the 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 during evaluation

[0281] Experimental results

[0282] Animal death situation

[0283] As shown in Table 9, the mortality rate of the mice in the model control group was 50%, and the mortality rate of the mice in the other groups was 0.

[0284] Table 9 Statistics of the number of surviving animals and mortality rates in each group

[0285]

[0286]

[0287] Effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the body weight of H22 hepatoma-bearing mice

[0288] As shown in Table 10, compared with the model control group, the body weight of mice in the cisplatin group was significantly decreased on D6, D12 - D22; the body weight of mice in the cisplatin + GLP-3 low-dose group was significantly decreased on D6 - D22; the body weight of mice in the cisplatin + GLP-3 high-dose group was significantly decreased on D3 - D22.

[0289] Compared with the cisplatin group, the body weight of mice in the cisplatin + GLP-3 low-dose group was significantly decreased on D3, D6, D12 - D22; the body weight of mice in the cisplatin + GLP-3 high-dose group was significantly decreased on D0 - D22.

[0290] Table 10 Effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the body weight of H22 hepatoma-bearing mice

[0291]

[0292] 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.

[0293] Effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the tumor volume of H22 hepatoma-bearing mice

[0294] As shown in Table 11, compared with the model control group, the tumor volume in the cisplatin group was significantly decreased from D9 - D22; the tumor volumes in the cisplatin + GLP-3 low-dose group and the cisplatin + GLP-3 high-dose group were significantly decreased on D3, D9 - D22.

[0295] Compared with the cisplatin group, the tumor volumes in the cisplatin + GLP-3 low-dose group and high-dose group were both significantly decreased from D9 - D22.

[0296] Table 11 Effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the tumor volume of H22 hepatoma-bearing mice

[0297]

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

[0299] Effect of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the relative tumor inhibition rate of H22 hepatoma-bearing mice

[0300] As shown in Table 12 and Table 13, compared with the model control group, the relative tumor inhibition rates of the cisplatin group from D9 to D22 were above 50%, being 53.1%, 64.4%, 73.1%, 71.6%, and 75.0% respectively; those of the cisplatin + low-dose GLP-3 group from D9 to D22 were above 70%, being 72.1%, 75.9%, 84.2%, 81.7%, and 80.4% respectively; those of the cisplatin + high-dose GLP-3 group from D9 to D22 were above 75%, being 77.6%, 83.4%, 89.2%, 90.4%, and 88.0% respectively.

[0301] Compared with the cisplatin group, the relative tumor inhibition rates of the cisplatin + low-dose GLP-3 group from D9 to D22 were 40.5%, 32.3%, 41.2%, 35.7%, and 21.4% (P < 0.05), and those of the cisplatin + high-dose GLP-3 group from D9 to D22 were above 50%, being 52.3%, 53.3%, 59.9%, 66.2%, and 51.9% (P < 0.05).

[0302] Table 12 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the relative tumor inhibition rate of H22 hepatoma-bearing mice (compared with the model control group)

[0303]

[0304] Note: Compared with the model control group, + P < 0.05.

[0305] Table 13 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the relative tumor inhibition rate of H22 hepatoma-bearing mice (compared with the cisplatin group)

[0306]

[0307] Note: Compared with the cisplatin group, # P < 0.05.

[0308] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on the organ index and tumor growth inhibition rate of H22 hepatoma-bearing mice

[0309] As shown in Table 14, compared with the model group, the tumor index, spleen index, and thymus index in the cisplatin group, cisplatin + GLP-3 low-dose group, and cisplatin + GLP-3 high-dose group were significantly decreased. Compared with the cisplatin group, the tumor index in the cisplatin + GLP-3 high-dose group was significantly decreased, and the spleen indexes in the cisplatin + GLP-3 low-dose group and cisplatin + GLP-3 high-dose group were significantly decreased. Compared with the normal group, the spleen index in the model control group was significantly increased; the thymus indexes in the cisplatin group, cisplatin + GLP-3 low-dose group, and cisplatin + GLP-3 high-dose group were significantly decreased.

[0310] Compared with the model control group, the tumor growth inhibition rate in the cisplatin group was 75.7%, and those in the cisplatin + GLP-3 low-dose group and high-dose group were 82.8% and 85.0% respectively. Compared with the cisplatin group, the tumor growth inhibition rates in the cisplatin + GLP-3 low-dose group and high-dose group were 29.4% and 38.4% respectively.

[0311] Table 14 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on organ indexes and tumor growth inhibition rate of H22 hepatoma-bearing mice

[0312]

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

[0314] As Figures 31 - 34 shown, the pictures of tumor-bearing mice are as follows: 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

[0315] As Figures 35 - 38 shown, the pictures of tumors of tumor-bearing mice are as follows: 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

[0316] Conclusion

[0317] Ganoderma lucidum polysaccharide GLP-3 combined with cisplatin can significantly inhibit the growth of tumors in H22 hepatoma-bearing mice and has a significant synergistic effect.

[0318] Experiment and data on the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on H22 orthotopic liver cancer mice

[0319] Purpose of the experiment

[0320] In this experiment, C57 mice were inoculated with H22 hepatoma cells under the right axilla to prepare mice with orthotopic tumors, and the anti-tumor effects of Ganoderma lucidum polysaccharide GLP-3 combined with chemotherapy on mice with H22 orthotopic liver cancer were studied to provide experimental basis for its clinical research.

[0321] Experimental materials

[0322] Test articles

[0323] Ganoderma lucidum polysaccharide GLP-3, batch number: ALM20200518A1, provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0324] Positive control articles

[0325] Kanglaite Soft Capsules, batch number: 20211009, Zhejiang Kanglaite Pharmaceutical Co., Ltd.; Cisplatin Injection, batch number: 601211204, Jiangsu Hansoh Pharmaceutical Group Co., Ltd.

[0326] Experimental animals

[0327] 100 SPF-grade male C57 mice, weighing 12 - 15 g, provided by Hunan Slack Jingda Experimental Animal Co., Ltd., production license number of experimental animals: SCXK(Xiang)2019 - 0004, quality certificate numbers of experimental animals: No430727221101898084, No430727221101898112, raised in Area D of the barrier environment animal laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd., license number for use of experimental animals: SYXK(Xiang)2020 - 0015.

[0328] Main reagents

[0329] 0.9% Sodium Chloride Injection, batch number: 21071401C, product of Hunan Kangyuan Pharmaceutical Co., Ltd.; Diluent for veterinary blood cell analysis, 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 products of Wako Pure Chemical Industries, Ltd.

[0330] Main instruments

[0331] AR223CN electronic balance, Ohaus Instruments (Changzhou) Co., Ltd.; LABOSPECT003 automatic biochemical analyzer, Hitachi, Japan; AniView100 multi-mode in vivo animal imaging system, ANDOR product; TDZ5-WS desktop multi-tube automatic balancing centrifuge, Hunan Kaida Industrial Development Co., Ltd.; ME2002E electronic balance, Shimadzu, Japan; flow cytometry instrument, BD product; ASP200S full-automatic tissue dehydrator, ASP300S full-automatic tissue dehydrator, TP1020 full-automatic dehydrator, HI1210 spreading machine, HI1220 baking machine, RM2235 paraffin slicer, EG1150H+C tissue embedding machine, AutoStainer XL automatic slide stainer + CV5030 automatic coverslipper, BX43 biological microscope + MD50 digital imaging system, CX31 biological microscope, Leica, Germany.

[0332] Experimental methods

[0333] Hepatocarcinoma (H22) cells labeled with Luc fluorescence at a concentration of 1×10 7 cells / mL were first inoculated into the peritoneal cavity of 8 male C57 mice. When ascites appeared in the mice, the ascites was aseptically aspirated, washed with HBSS buffer, centrifuged to discard the supernatant, stained with trypan blue, and the ascites cells were counted under a microscope. The cell number was adjusted to 1×10 13 cells / mL and inoculated into the right liver lobe of 80 male C57 mice to prepare orthotopic tumor-bearing mice, with an inoculation volume of 10 μL / mouse. One week later, the tumor formation in the mouse liver was detected using a small animal in vivo imager. The mice were randomly grouped according to tumor size as follows: model control group, radiotherapy group (2 Gy / d), 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 + GLP-3 low-dose group (2 Gy + 130 mg / kg), radiotherapy + GLP-3 high-dose group (2 Gy + 1170 mg / kg), with 10 mice in each group. Another 10 mice were used as the normal control group. Except for the normal control group, the remaining animals received radiotherapy. The radiotherapy animals were irradiated using an animal radiotherapy instrument. The animals were anesthetized, placed in a self-made lead apron, and the tumor tissue was exposed for radiotherapy. 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 injected with cisplatin intraperitoneally. The remaining groups of mice were given the corresponding medicinal solutions, with the administration volumes being 20 mL / kg (by gavage) and 10 mL / kg (by intraperitoneal injection), once a day for 14 consecutive days. After the last administration, blood was collected from the orbital cavity to detect blood WBC, RBC, liver and kidney function indices (ALT, AST, BUN, CRE), and CD3 + / CD4 + and CD3 + / CD8 + After weighing the spleen, thymus, and tumor, the organ coefficients were calculated, and histopathological examinations were performed on the liver, spleen, and thymus.

[0334] Dose Design

[0335] Based on the results of previous experiments, Ganoderma lucidum polysaccharide GLP-3 was designed with a low dose of 130 mg / kg and a high dose of 1170 mg / kg. The corresponding drugs were administered as shown in Table 15.

[0336] The clinical intended dosage of Kanglaite Soft Capsules is 0.45 g / capsule, 6 capsules / time, 4 times / day, which is 10.8 g / day. Converted to the equivalent dose for mice according to body surface area, it is 10.8 g / day * 0.0026 / 0.02 kg = 1404 mg / kg. This experiment was conducted at an equal multiple of the clinically intended dosage.

[0337] In this experiment, the radiotherapy intensity was designed at an equal multiple of the clinically intended dosage, which is 2 Gy / day.

[0338] Table 15 Experiment Grouping and Dose Design

[0339]

[0340]

[0341] Detection Indexes

[0342] Efficacy Indexes

[0343] Animal survival rate and general condition: Weigh the animals once a week and record the death situation.

[0344] Tumor volume detection: The changes in tumor volume of each group were detected weekly using small animal in vivo imaging technology.

[0345] Hematological detection: Detection of blood routine (WBC, RBC) and biochemical (liver and kidney function) indexes after the last administration.

[0346] Immune organs: Weigh the thymus, spleen, tumor, and liver, and calculate the organ coefficients. Organ coefficient (%) = organ weight / body weight after fasting × 100%.

[0347] CD4 + and CD8 + Content detection: After the last administration, flow cytometry was used to detect the lymphocyte subsets CD3 + / CD4 + and CD3 + / CD8 + content.

[0348] Data processing and statistical analysis

[0349] The significant figures of the data in this experiment were rounded according to the rule of rounding up or down, and statistical analysis was carried out according to the regulations of the central SOP. The software used for statistics

[0350] was SPSS. Measurement data were expressed as mean ± standard deviation and the normality and homogeneity of variance were tested by Leven’s test. If there was no statistical significance (P>0.05), one-way analysis of variance (ANOVA) was used for statistical analysis. If ANOVA had statistical significance (P≤0.05), LSD test (parametric method) was used for comparative analysis. If the variances were heterogeneous (P≤0.05), Kruskal-Wallis test was used. If Kruskal-Wallis test had statistical significance (P≤0.05), Dunnett’s Test (non-parametric method) was used for comparative analysis. The statistical results were based on the test limit of α = 0.05, where P≤0.05 indicated statistical significance and P≤0.01 indicated very significant difference in the tested differences

[0351] Experimental results

[0352] Animal death situation

[0353] In the model control group after administration, mice 2M07 / 2M08 and 2M05 died on days D4 and D10 respectively; in the radiotherapy group, mice 3M02 / 3M10, 3M07, 3M09 / 3M01, and 3M05 died on days D3, D4, D5, and D10 respectively; in the radiotherapy + cisplatin group, mice 4M03 / 4M09, 4M06, 4M10, 4M07, and 4M04 died on days D4, D6, D7, D8, and D13 respectively; in the Kanglaite soft capsule group, mice 5M03, 5M07, and 5M08 died on days D8, D10, and D14 respectively; in the radiotherapy + Kanglaite soft capsule group, mice 6M10, 6M08 / 6M02, 6M05, 6M06, and 6M01 / 6M07 died on days D7, D8, D10, D13, and D14 respectively; in the radiotherapy + GLP-3 low-dose group, mouse 7M01 died on day D8; in the radiotherapy + GLP-3 high-dose group, mice 8M03, 8M08, 8M06, and 8M02 died on days D9, D10, D13, and D14 respectively

[0354] The mortality rates of each group were 0%, 30%, 60%, 60%, 30%, 70%, 10%, and 50% respectively

[0355] Table 16 Statistics of the number of surviving animals and mortality rates in each group

[0356]

[0357] Effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on the body weight of mice with H22 orthotopic liver cancer

[0358] 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 the 0th, 1st, and 2nd 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 GLP-3 low-dose group was significantly decreased at the 1st and 2nd weeks after administration (P≤0.05 or P≤0.01); the body weight of mice in the radiotherapy combined with GLP-3 high-dose group was significantly decreased at the 1st 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 the 1st and 2nd 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 GLP-3 low- and high-dose groups and the Kanglaite soft capsule group was significantly increased at the 1st and 2nd weeks after administration (P≤0.05 or P≤0.01). There was no significant difference in the remaining drug administration groups.

[0359] Table 17 Effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on the body weight of mice with H22 orthotopic liver cancer (Mean±SEM)

[0360]

[0361] 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.

[0362] Effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on the tumor of mice with H22 orthotopic liver cancer

[0363] As Figure 39 、 40 shown in Table 18, compared with the normal control group, the tumor of mice in the model control group was significantly increased (P≤0.01); compared with the model control group, the tumors of mice in the radiotherapy combined with GLP-3 low- and high-dose groups and the radiotherapy combined with cisplatin group were significantly decreased at the 2nd week after administration (P≤0.05 or P≤0.01). Compared with the Kanglaite soft capsule group, the tumor of mice in the radiotherapy combined with GLP-3 high-dose group was significantly decreased at the 2nd week after administration (P≤0.05). There was no significant difference in the remaining groups.

[0364] As Figure 39As shown, 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.

[0365] As Figure 40 shown, 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 + low-dose GLP-3 group, 8: radiotherapy + high-dose GLP-3 group.

[0366] Table 18 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on tumors in mice with H22 orthotopic liver cancer (Mean±SEM)

[0367]

[0368]

[0369] 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 the radiotherapy + cisplatin group, & P≤0.05; compared with the Kanglaite soft capsule group, ★ P≤0.05; compared with the radiotherapy + Kanglaite soft capsule group, ■ P≤0.05.

[0370] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on organ coefficients and tumor growth inhibition rate in mice with H22 orthotopic liver cancer

[0371] As shown in Table 19, compared with the normal control group, the organ coefficients of the spleen and liver tissues in the model control group were significantly increased (P≤0.01), 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, Kanglaite soft capsule group, cisplatin group, and radiotherapy 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 group and cisplatin group 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 coefficients of mice in the radiotherapy combined with low- and high-dose GLP-3 groups were significantly decreased (P≤0.01). Compared with the radiotherapy + Kanglaite soft capsule group, there were no significant differences among the groups.

[0372] Table 19 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on organ coefficients of H22 orthotopic liver cancer mice (Mean±SEM)

[0373]

[0374] 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 the radiotherapy + cisplatin group, & P≤0.05, && P≤0.01; compared with the radiotherapy + Kanglaite soft capsule group, ■ P≤0.05.

[0375] Effects of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on blood biochemical indexes of H22 orthotopic liver cancer mice

[0376] As shown in Table 20, compared with the normal control group, the blood WBC, AST, BUN, and CRE of the model control group mice were significantly increased (P≤0.05 or P≤0.01). Compared with the model control group, the blood WBC, AST, BUN, and CRE of the radiotherapy combined with GLP-3 low-dose group, cisplatin group, Kanglaite soft capsule group, and radiotherapy group mice were significantly decreased (P≤0.05 or P≤0.01); the blood WBC, BUN, and CRE of the radiotherapy combined with GLP-3 high-dose group mice were significantly decreased (P≤0.05 or P≤0.01); the blood WBC and CRE of the Kanglaite soft capsule group mice were significantly decreased (P≤0.05 or P≤0.01). Compared with the radiotherapy group, the blood CRE of the radiotherapy combined with GLP-3 low- and high-dose group mice was significantly decreased (P≤0.05); the blood WBC, AST, and BUN of the Kanglaite soft capsule group mice were significantly increased (P≤0.05 or P≤0.01), and the CRE was significantly decreased (P≤0.05). Compared with the radiotherapy + cisplatin group, the blood WBC, AST, and BUN of the Kanglaite soft capsule group mice were significantly increased (P≤0.05 or P≤0.01). Compared with the Kanglaite soft capsule group, the blood AST and BUN of the radiotherapy combined with GLP-3 low-dose group mice were significantly decreased (P≤0.05 or P≤0.01); the blood BUN of the radiotherapy combined with GLP-3 high-dose group and Kanglaite soft capsule group mice was significantly decreased (P≤0.05 or P≤0.01). There were no significant differences in the remaining groups.

[0377] Table 20 Effects of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on blood biochemical indexes of H22 orthotopic liver cancer mice (Mean±SEM)

[0378]

[0379]

[0380] 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 the Kanglaite soft capsule group, ★ P ≤ 0.05, ★★ P ≤ 0.01.

[0381] Effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on CD3 + / CD4 + and CD3 + / CD8 + in blood of mice with H22 orthotopic liver cancer

[0382] As shown in Table 21, compared with the normal control group, the levels of CD3+ / CD4+ and CD3+ / CD8+ in the blood of mice in the model control group showed an increasing trend, but there was no significant difference. Compared with the model control group, there was no significant difference in each drug administration group. Compared with the radiotherapy + cisplatin group, in the radiotherapy combined with low-dose GLP-3 group and the Kanglaite soft capsule group, the level of CD3+ / CD8+ in the blood of mice in the Kanglaite soft capsule group was significantly decreased (P ≤ 0.05 or P ≤ 0.01). There was no significant difference in the remaining groups.

[0383] Table 21 Effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy on CD3 + / CD4 + and CD3 + / CD8 + in blood of mice with H22 orthotopic liver cancer (Mean±SEM)

[0384]

[0385] Note: Compared with the model control group, *P ≤ 0.05; compared with the radiotherapy + cisplatin group, & P ≤ 0.05, && P ≤ 0.01.

[0386] Such as Figure 41 and 42As shown in the results of 43, a large number of H22 hepatoma cells infiltrated and necrosis occurred in the livers of mice in the model control group, the number of sinusoidal cells in the liver increased, and inflammatory cell infiltration appeared; extramedullary hematopoiesis in the spleen increased significantly, the red pulp was diffuse, and hepatoma cells infiltrated in the thymus, and the number of lymphocytes in the cortex / medulla decreased. A large number of H22 hepatoma cells infiltrated and necrosis occurred in the livers of mice in the radiotherapy group and the radiotherapy + cisplatin group, extramedullary hematopoiesis in the red pulp of the spleen increased, the number of white pulp cells decreased, and the number of lymphocytes in the thymus decreased. After administration of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy, the degree of infiltration and necrosis of hepatoma cells in the livers of each group decreased, extramedullary hematopoiesis in the spleen increased, the number of plasma cells in the white pulp increased, and the degree of thymus lesions decreased.

[0387] As Figure 41 , 42 , 43 shown, the corresponding groups were: 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

[0388] Conclusion

[0389] Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy can significantly inhibit the growth of tumors in mice with H22 orthotopic liver cancer and has an obvious synergistic effect.

[0390] Discussion and Summary

[0391] Liver cancer is a malignant tumor with extremely high mortality. At present, surgical resection combined with chemotherapy intervention and radiotherapy are mostly used to delay symptoms, and it is very difficult to completely cure. Liver cancer is insensitive to chemotherapy. Especially for patients with advanced liver cancer, there is still no reliable evidence to prove that systemic chemotherapy can improve the overall survival of advanced liver cancer.

[0392] The results of this experiment showed that the tumor volume of 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 decreased during the tumorigenesis process in mice in the model control group. After the last administration, Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy could significantly inhibit the growth of tumors in mice, the thymus index and spleen index decreased significantly, the number of red blood cells in the blood of mice decreased significantly, and the content of liver and kidney function indexes decreased significantly. CD4 + T cells and CD8 + T cells mediate tumor immune responses, among which CD8 + T cells are the main effector cells of tumor immunity. The results showed that the CD3 + / CD4 + , CD3 + / CD8 +The content decreased significantly, indicating that Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy can protect immune organs, enhance the body's autoimmune function, and thus play a role in 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 capsule combined with radiotherapy, the tumor, spleen coefficient, and thymus coefficient were significantly reduced when Ganoderma lucidum polysaccharide GLP-3 was combined with radiotherapy. The results of this experiment showed that the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-3 combined with radiotherapy was stronger than that of Kanglaite soft capsule combined with radiotherapy.

[0393] 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 an obvious synergistic effect.

[0394] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Ganoderma lucidum polysaccharide GLP-3, characterized in that, Molecular structural formula of the Ganoderma lucidum polysaccharide GLP-3 ; Molecular formula: (C 66 H 110 O 55 ) n The main chain of the Ganoderma lucidum polysaccharide GLP-3 is a 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 respectively connected to the main chain through the o-3 of →3,6)-β-D-Glcp-(1→ and the o-4 of →4,6)-β-D-Glcp-(1→. The molecular simplified formula is: , 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 the Ganoderma lucidum polysaccharide GLP-3 according to claim 1 or 2, characterized in that, This method comprises the following steps: S1. Remove the dust from Ganoderma lucidum and dry it, then crush it to make Ganoderma lucidum powder; S2. Place the crushed Ganoderma lucidum powder in a sealed container, mix it with water and heat, and fully blend the Ganoderma lucidum powder and water into a medicinal juice solution under high temperature and high pressure; S3. Use membrane concentration technology to separate the medicinal juice solution to obtain a concentrated solution containing active pharmaceutical ingredients and incompletely blended medicinal residues; S4. Mix the concentrated solution containing active pharmaceutical ingredients with pure water to form an aqueous solution with a preset concentration, and after multiple column chromatography separations and concentration and freeze-drying, obtain Ganoderma lucidum polysaccharide GLP-3 with active pharmaceutical ingredients.

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

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

6. The method for extracting Ganoderma lucidum polysaccharide GLP-3 according to claim 5, characterized in that, In step S4, the concentrated solution containing active pharmaceutical ingredients is formulated with pure water at a concentration ratio of 1:2 - 1:

5.

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

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

9. The method for extracting Ganoderma lucidum polysaccharide GLP-3 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, 125 °C, 130 °C, 135 °C, 140 °C, 145 °C, 155 °C, 160 °C, 165 °C or 170 °C, and the boiling time is 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h or 6 h. The Ganoderma lucidum powder and water in the sealed container are fully blended into a mixed medicinal liquid under high temperature and high pressure.

10. The method for extracting Ganoderma lucidum polysaccharide GLP-3 according to claim 4, characterized in that, In step S2, the mixed liquid in the sealed container is heated to 105 °C, 110 °C, 115 °C, 125 °C, 130 °C, 135 °C, 140 °C, 145 °C, 155 °C, 160 °C, 165 °C, 170 °C, 175 °C, 180 °C, 185 °C, 190 °C, 195 °C or 200 °C, and the boiling time is 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h or 6 h. The Ganoderma lucidum powder and water in the sealed container are fully blended into a mixed medicinal liquid under high temperature and high pressure.

11. The application of the Ganoderma lucidum polysaccharide GLP-3 and cisplatin according to claim 1 or 2 in the preparation of anti-lung cancer and anti-liver cancer pharmaceutical products, characterized in that, When the Ganoderma lucidum polysaccharide GLP-3 and the chemotherapeutic drug cisplatin are used in combination, they can eliminate the toxic and side effects caused by the chemotherapeutic drug to the human body and have an effect on the control, reduction of tumor masses, and reduction and elimination of tumor cells.

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