A polysaccharide compound with clear molecular structure and capable of eliminating toxic side effects of chemotherapy drugs

Abstract

The present application is suitable for the technical field of plant extraction and separation, and provides a polysaccharide compound with clear molecular structure, which is suitable for treating patients with advanced cancer (surgical conditions have been lost, survival time is only three to six months, and chemotherapy conditions are still available), comprising: S1, drying and crushing Ganoderma lucidum; S2, mixing with water in a sealed container to extract effective components under high temperature and high pressure; S3, removing residues from the concentrated liquid with effective components; and S4, preparing the residue-removed concentrated liquid into a certain concentration to obtain polysaccharide with effective components through column chromatography. The polysaccharide with effective components has good water-soluble performance, is easy to be absorbed by the human body, has antitumor efficacy, and has efficacy in preventing the occurrence of tumors in humans, especially when used in combination with chemotherapy drugs, the toxic and side effects of the chemotherapy drugs on the human body can be eliminated, and the control, reduction, and elimination of tumor cells are all supported by corresponding experimental data.

Description

Technical Field

[0001] This invention belongs to the field of extraction and separation technology improvement, and particularly relates to a method for extracting Ganoderma lucidum polysaccharide GLP-1 and its application. Background Technology

[0002] Reishi mushroom is a type of fungus with a long history of medicinal use in China and Japan. Reishi contains numerous active ingredients with a complex composition; over 150 compounds have been isolated from it, primarily polysaccharides, triterpenoids, sterols, alkaloids, furan derivatives, amino polypeptides, and inorganic elements. Different geographical locations (longitude and latitude), different seeds, different growth environments, and varying temperatures, humidity, and light intensity all significantly influence the content, proportion, and presence of the medicinal components mentioned in this invention within Reishi. Summary of the Invention

[0003] The purpose of this invention is to provide an improved method and application for extracting polysaccharide compounds with clear molecular structures and medicinal functions from Ganoderma lucidum raw materials.

[0004] This invention aims to solve the current international challenge of treating advanced cancer patients, which is defined as those who have lost the ability to undergo surgery, have a life expectancy of only three to six months, and are still eligible for chemotherapy. The treatment challenge is to help advanced cancer patients regain their appetite as quickly as possible (generally within two to three weeks), shrink or stop the growth of tumors, and, when used in combination with chemotherapy drugs, to largely eliminate the toxic side effects of chemotherapy drugs. Long-term use in combination with chemotherapy drugs can achieve the goal of basically eliminating cancer cells or controlling the number of cancer cells within a safe range for high-quality human survival.

[0005] The active ingredient referred to in this invention also has an important function: it can prevent the mutation of normal human cells and prevent the formation of cancer cells.

[0006] The active pharmaceutical ingredient referred to in this invention also has an important function: when used in combination with chemotherapy drugs, it exhibits exceptionally good therapeutic effects on lung cancer, liver cancer, and breast cancer, demonstrating a certain degree of broad-spectrum efficacy.

[0007] This invention is achieved by a method for extracting Ganoderma lucidum polysaccharide GLP-1, which includes the following steps:

[0008] S1. After removing dust and drying the Ganoderma lucidum, it is crushed to make Ganoderma lucidum powder;

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

[0010] S3. Using membrane concentration technology, the medicinal juice solution is separated to obtain a concentrated solution with medicinal components and incompletely blended medicinal residue;

[0011] S4. The concentrated solution containing the active ingredient is mixed with pure water to form an aqueous solution of a preset concentration. The solution is then subjected to multiple column chromatography separation techniques to obtain Ganoderma lucidum polysaccharide GLP-1 containing the active ingredient.

[0012] A further technical solution of the present invention is: in step S2, the mixture of Ganoderma lucidum powder and water in the sealed container is thoroughly stirred and then heated to 105-200°C, and the boiling time is maintained for 2-6 hours. As the heating temperature rises, the internal pressure of the sealed container gradually increases, forming a high temperature and high pressure environment in the sealed container.

[0013] A further technical solution of the present invention is: in step S2, the mixture of Ganoderma lucidum powder and water in the sealed container is heated to 105-170°C at a high temperature and boiled for 3-6 hours. As the heating temperature rises, the internal pressure of the sealed container gradually increases, forming a high temperature and high pressure environment in the sealed container.

[0014] A further technical solution of the present invention is: in step S4, the concentrated solution containing the active pharmaceutical ingredient is prepared with pure water to a concentration ratio of 1:2-1:5.

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

[0016] A further technical solution of the present invention is: in step S1, the Ganoderma lucidum is rinsed with clean water to remove surface dust, dried at 105°C, and the dried Ganoderma lucidum is crushed, and the crushed Ganoderma lucidum powder is larger than 60 mesh.

[0017] A further technical solution of the present invention is: in step S2, the mixed liquid in the sealed container is heated to a high temperature of 105℃, 110℃, 115℃, 120℃, 125℃, 130℃, 135℃, 140℃, 145℃, 150℃, 155℃, 160℃, 165℃, 170℃, 175℃, 180℃, 185℃, 190℃, 195℃ or 200℃, and the boiling time is 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h. As the heating temperature increases, the internal pressure of the sealed container gradually increases, forming a high temperature and high pressure environment in the sealed container.

[0018] Another object of the present invention is to provide a Ganoderma lucidum polysaccharide GLP-1, wherein the structural formula of the Ganoderma lucidum polysaccharide GLP-1 is... Molecular formula (C 60 H 100 O 50)n, where n = 17-25.

[0019] A further technical solution of the present invention is that n is 17, 18, 19, 20, 21, 22, 23, 24 or 25.

[0020] Another objective of this invention is to provide an application of Ganoderma lucidum polysaccharide GLP-1, which 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. In particular, 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, as well as reducing and eliminating tumor cells.

[0021] The beneficial effects of this invention are: the extraction process is simple, the polysaccharide extraction rate is high, the production cost is low, and it is easy to industrialize; Ganoderma lucidum polysaccharide GLP-1 has good solubility and is easily absorbed by the human body, thereby exerting its anti-tumor, antioxidant, and anti-aging effects. Attached Figure Description

[0022] Figure 1 This is a flowchart of the method for extracting Ganoderma lucidum polysaccharide GLP-1 provided in the embodiments of the present invention.

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

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

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

[0026] Figure 5 This is a schematic diagram of the molecular weight spectrum of the sample provided in the embodiment of the present invention.

[0027] Figure 6 This is a schematic diagram of the ion chromatogram of the mixed standard 16 sugar provided in the embodiment of the present invention. Figure 1 .

[0028] Figure 7 This is a schematic diagram of the ion chromatogram of the mixed standard 16 sugar provided in the embodiment of the present invention. Figure 2 .

[0029] Figure 8 This is a GCMS chromatogram of the sample (PMAA) provided in an embodiment of the present invention.

[0030] Figure 9 This is a schematic diagram of the analysis results of polysaccharide methylated sugar alcohol acetyl ester (PMAA) provided in the embodiments of the present invention. Figure 1 .

[0031] Figure 10 This is a schematic diagram of the analysis results of polysaccharide methylated sugar alcohol acetyl ester (PMAA) provided in the embodiments of the present invention. Figure 2 .

[0032] Figure 11 This is a schematic diagram of the analysis results of polysaccharide methylated sugar alcohol acetyl ester (PMAA) provided in the embodiments of the present invention. Figure 3 .

[0033] Figure 12 This is a schematic diagram of the analysis results of polysaccharide methylated sugar alcohol acetyl ester (PMAA) provided in the embodiments of the present invention. Figure 4 .

[0034] Figure 13 This is a schematic diagram of the hydrogen spectrum provided in an embodiment of the present invention.

[0035] Figure 14 This is a schematic diagram of carbon spectrum provided in an embodiment of the present invention.

[0036] Figure 15 This is a schematic diagram of the Dept135 spectrum provided in an embodiment of the present invention.

[0037] Figure 16 This is a schematic diagram of HH-COSY provided in an embodiment of the present invention.

[0038] Figure 17 This is the HSQC diagram provided in the embodiment of the present invention.

[0039] Figure 18 This is the HMBC map provided in the embodiments of the present invention.

[0040] Figure 19 This is a schematic diagram of NOESY provided in an embodiment of the present invention.

[0041] Figure 20 This is a schematic diagram showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the tumor volume of Lewis cell tumor-bearing mice, as provided in the embodiments of the present invention.

[0042] Figure 21 This is a schematic diagram (×200) showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the tumor pathology of Lewis cell tumor-bearing mice provided in the embodiments of the present invention.

[0043] Figure 22 This is a schematic diagram (×200) showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the kidney pathology of Lewis cell tumor-bearing mice provided in the embodiments of the present invention.

[0044] Figure 23This is a schematic diagram (×200) showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on gastric pathology in Lewis cell tumor-bearing mice provided in this embodiment of the invention.

[0045] Figure 24 This is a schematic diagram (×200) showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the spleen pathology of Lewis cell tumor-bearing mice provided in this embodiment of the invention.

[0046] Figure 25 This is a schematic diagram illustrating the effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on tumor volume in Lewis cell tumor-bearing mice, as provided in this embodiment of the invention.

[0047] Figure 26 This is a schematic diagram (×200) showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on the pathological changes of thymus tissue in Lewis cell tumor-bearing mice provided in this embodiment of the invention.

[0048] Figure 27 This is a schematic diagram (×200) showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on the pathological characteristics of spleen tissue in Lewis cell tumor-bearing mice provided in this embodiment of the invention.

[0049] Figure 28 This is a schematic diagram (×200) showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on the nasal tissue pathology of Lewis cell tumor-bearing mice provided in this embodiment of the invention.

[0050] Figure 29 This is a schematic diagram (×200) showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on the pathological condition of gastric tissue in Lewis cell tumor-bearing mice provided in this embodiment of the invention.

[0051] Figure 30 This is a schematic diagram (×200) showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on the pathological characteristics of kidney tissue in Lewis cell tumor-bearing mice provided in this embodiment of the invention.

[0052] Figure 31 , 32 33 and 34 are schematic diagrams of the effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on H22 liver cancer-bearing mice provided in the embodiments of the present invention.

[0053] Figure 35 , 36 37 and 38 are schematic diagrams of the tumor appearance of the effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on H22 liver cancer-bearing mice provided in the embodiments of the present invention.

[0054] Figure 39 , 4041 and 42 are schematic diagrams of the effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on 4T1 breast cancer-bearing mice provided in the embodiments of the present invention.

[0055] Figure 43 , 44 45 and 46 are schematic diagrams of the tumor appearance of the effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on 4T1 breast cancer-bearing mice provided in the embodiments of the present invention.

[0056] Figure 47 , 48 Figures 49, 50, 51, and 52 are schematic diagrams illustrating the effects of Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin on LLC lung cancer cells according to embodiments of the present invention. Detailed Implementation

[0057] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0058] like Figure 1 The flowchart of the method for extracting Ganoderma lucidum polysaccharide GLP-1 provided by the present invention is shown below, and its details are as follows:

[0059] Step S1: Wash the harvested Ganoderma lucidum or the Ganoderma lucidum that has undergone preliminary simple processing with clean water on the cleaning equipment to remove the floating dust on its surface. After removing the floating dust, transfer the Ganoderma lucidum to the drying equipment for drying at a drying temperature of 105℃. Place the dried Ganoderma lucidum on the crusher to crush it into Ganoderma lucidum powder. The Ganoderma lucidum powder is sieved. If the Ganoderma lucidum powder particles are larger than 60 mesh, they are transferred to the next process. If the Ganoderma lucidum powder particles are smaller than 60 mesh, they are returned to the crushing mechanism for further crushing. Repeat the process multiple times until the crushed Ganoderma lucidum powder particles meet the specified requirements.

[0060] Step S2: Mix the qualified Ganoderma lucidum powder with pure water and place it in a sealed container. Heat the sealed container at a high temperature. During the heating process, the internal pressure of the sealed container gradually increases with the rise in temperature, creating a high-temperature and high-pressure environment inside the sealed container. Under this environment, it is easier for the Ganoderma lucidum powder and water to fully blend into a mixed liquid. The sealed container is heated to a temperature between 105℃ and 200℃ for 2-6 hours, preferably between 105℃ and 170℃. The boiling time is 3-6 hours, more preferably high temperature heating to 105℃, 110℃, 115℃, 120℃, 125℃, 130℃, 135℃, 140℃, 145℃, 150℃, 155℃, 160℃, 165℃, 170℃, 175℃, 180℃, 185℃, 190℃, 195℃ or 200℃, and 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 mixture is fully blended.

[0061] Step S3: Centrifuge the extracted aqueous solution containing the medicinal components to remove residue, and then concentrate the centrifuged liquid through a membrane to obtain a concentrated solution.

[0062] Step S4: Prepare a concentrated solution containing medicinal components to a certain concentration and separate it by column chromatography to obtain Ganoderma lucidum polysaccharide GLP-1 containing medicinal components.

[0063] This method has a simple extraction process, high polysaccharide extraction rate, low production cost, and is easy to operate.

[0064] Another object of the present invention is to provide a Ganoderma lucidum polysaccharide GLP-1, wherein the structural formula of the Ganoderma lucidum polysaccharide GLP-1 is... Molecular formula (C 60 H 100 O 50 )n, where n = 17-25.

[0065] n is 17, 18, 19, 20, 21, 22, 23, 24 or 25.

[0066] After obtaining the Ganoderma lucidum polysaccharide GLP-1 with the above structure, an experiment was conducted on it, and the results are reported below.

[0067] I. Molecular weight determination

[0068] 1. Experimental Objective

[0069] The molecular weight and purity of the polysaccharide were determined by HPGPC.

[0070] 2. Experimental Materials

[0071] 2.1 Instruments

[0072]

[0073] 2.2 Materials

[0074] reagents factory batch number Item number level Valid Date NaCl ACROS A0356762 139725000 ACROS 2022

[0075] 2.3 Standard Products

[0076]

[0077]

[0078] 3. Experimental Procedure

[0079] 3.1 Reagent Preparation

[0080] Reagent Name Preparation method Storage conditions Validity period 0.05M NaCl solution Precisely prepared, filtered through a 0.45μm membrane, and ultrasonically degassed for 10 minutes. RT one month

[0081] 3.2 Preparation of Sample and Standard Solutions

[0082] 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 through a 0.22 μm microporous membrane, and then transfer the sample to a 1.8 ml vial.

[0083] 3.3 Chromatographic Methods

[0084] Chromatographic column: BRT105-104-102 tandem gel column (8×300mm); mobile phase: 0.05M NaCl solution; flow rate: 0.6ml / min; column temperature: 40℃; injection volume: 20μl; detector: differential detector RI-10A.

[0085] 4. Experimental Results

[0086] like Figure 2-4 As shown, the calibration curves for lgMp-RT (peak molecular weight), lgMw-RT (weight-average molecular weight), and lgMn-RT (number-average molecular weight) were obtained.

[0087] The equation for the lgMp-RT correction curve is: y = -0.1802x + 11.661R² = 0.9928;

[0088] The equation of the lgMw-RT correction curve is: y = -0.1926x + 12.241R² = 0.9965;

[0089] The equation of the lgMn-RT correction curve is: y = -0.1783x + 11.506R² = 0.9911;

[0090] 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 below. Figure 5 As shown in the figure, the calculation results are shown in the table below.

[0091] SampleID RT(min) lgMp lgMw lgMn Mp Mw Mn Peak area ratio % 39.67 4.5 4.6 4.4 32544 39862 27092 100

[0092] The peak at 46.5 min is the mobile phase peak.

[0093] II. Monosaccharide Composition Determination Experiment

[0094] 1. Experimental Objective

[0095] Monosaccharide composition was determined using ion chromatography.

[0096] 2. Experimental Principle

[0097] Based on the electrochemical activity of carbohydrate molecules and their ionized state in strongly alkaline solutions, and considering that carbohydrate compounds are weak acids with pKa > 11, they exist partially or entirely as anions in high-pH eluents. By leveraging the differences in ion exchange efficiency caused by variations in the pKa of different carbohydrate compounds and the varying 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 molecule on the gold electrode surface is then detected.

[0098] 3. Experimental Materials

[0099] 3.1 Instruments

[0100]

[0101]

[0102] 3.2 Reagents

[0103] reagents factory batch number Item number level Trifluoroacetic acid ACROS A0356762 139725000 AR 50% sodium hydroxide solution Alfa Aesar Z21E036 33382 GR Sodium acetate ThermoFishe 191126 059326 GR

[0104] 3.3 Standard Products

[0105] Standard products factory batch number Storage conditions purity Mannose Borui Sugar Bio C17D9H77586 Store in an airtight container AR Rhamnose Borui Sugar Bio H10S9Z69863 Store in an airtight container AR Galacturonic acid Borui Sugar Bio K02A9B66077 Store in an airtight container AR Galactose Borui Sugar Bio E1927035 Store in an airtight container AR glucose Borui Sugar Bio Q18F10N80946 Store in an airtight container AR Glucuronic acid Borui Sugar Bio K14M10S82777 Store in an airtight container AR Arabic sugar Borui Sugar Bio S15A10G85850 Store in an airtight container AR Xylose Borui Sugar Bio A22S6X3606 Store in an airtight container AR Fucose Borui Sugar Bio X29D7Y27768 Store in an airtight container AR Glucosamine hydrochloride Borui Sugar Bio A22S6X3606 Store in an airtight container AR N-acetylglucosamine Borui Sugar Bio A21J8X40372 Store in an airtight container AR D-fructose Borui Sugar Bio J01J10R89818 Store in an airtight container AR D-ribose Borui Sugar Bio H26F10Z81556 Store in an airtight container AR Aminogalactoside Borui Sugar Bio B01J8S37079 Store in an airtight container AR L-guluronic acid Borui Sugar Bio S200115AG1 Store in an airtight container ≥98％ D-mannuronic acid Borui Sugar Bio S200108AM1 Store in an airtight container ≥98％

[0106] 4. Experimental Methods

[0107] 4.1 Reagent Preparation

[0108] Reagent Name Preparation method Storage conditions 15mM NaOH solution 2.4g 50% NaOH solution, 2L water RT 15mMNaOH&100mMNaOAC solution 1.2g 50% NaOH solution, 8.2g NaOAC, 1L water RT

[0109] 4.2 Preparation and Calculation Methods of Standard Solutions

[0110] Prepare a standard stock solution by taking 16 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosyl hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, and mannuronic acid).

[0111] Precisely prepare concentration standards from the standard solutions of each monosaccharide to form a mixed standard. Determine the mass of different monosaccharides using an absolute quantification method, and calculate the molar ratio based on the molar mass of the monosaccharides.

[0112] 4.3 Sample Preparation

[0113] Accurately weigh 5 mg of sample into 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. Pipette 50 μL of the solution into 950 μL of deionized water and centrifuge at 12000 rpm for 5 min. Collect the supernatant for IC analysis.

[0114] 4.4 Chromatographic Methods

[0115] Chromatographic column: Dionex Carbopac™ PA20 (3*150mm); Mobile phase: A: H2O; B: 15mM NaOH; C: 15mM NaOH & 100mM NaO; Flow rate: 0.3ml / min; Injection volume: 5μL; Column temperature: 30℃; Detector: Electrochemical detector.

[0116] 4.5 Standard Product Sequence

[0117] 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 Arabic sugar 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-acetylglucosamine 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

[0118] C(standard) / A(standard) = C(sample) / A(sample)

[0119] 5. Experimental Results

[0120] Mixed standard: Solvent peaks: Sodium hydroxide peak at 2.0 min, sodium acetate peak at 41 min. For example... Figure 6 As shown.

[0121] Name RT Mole ratio Fucose 5.959 0.000 galactosamine hydrochloride 10.817 0.000 Rhamnose 11.334 0.000 Arabic sugar 11.792 0.000 Glucosamine hydrochloride 13.384 0.000 Galactose 14.609 0.000 glucose 16.809 0.887 N-acetylglucosamine 18.55 0.000 Xylose 19.209 0.000 Mannose 19.825 0.000 fructose 22.259 0.000 Ribose 24.309 0.000 Galacturonic acid 44.592 0.000 guluronic acid 45.184 0.000 Glucuronic acid 47.317 0.113 Mannuronic acid 50.2 0.000

[0122] III. Experiment to determine polysaccharide linkage mode

[0123] 1. Experimental Objective

[0124] After derivatization such as methylation, the linkage mode of polysaccharide samples was determined by GC-MS.

[0125] 2. Experimental Materials

[0126] 2.1 Instruments

[0127] Instrument Name factory model Rotary evaporator Zhengzhou Great Wall Science & Industry Trade Co., Ltd. R-1001VN Nitrogen blowing device Lichen Technology UGC-24M Magnetic stirrer DLAB MS7-H550-Pro Vacuum drying oven Lichen Technology 101-1BS Gas Chromatography-Mass Spectrometry Agilent 6890-5973

[0128] 2.2 Reagents

[0129] reagents factory batch number Item number level Trifluoroacetic acid ACROS A0356762 139725000 AR Iodomethane Adamas P1345479 01111630 AR Sodium borohydride Aldrich MKCD7945 205591 AR Ethyl acetate Wokai 08050003 40065982 AR Acetic anhydride Shanghai test 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 Shanghai test 20150429 10019718 AR dimethyl sulfoxide Adamas P1265087 759270 AR Sodium hydride Adamas P1306059 81778A AR Methylation kit Borui Saccharide BRT-2020JJH BRT-JJH AR

[0130] 3. Experimental Methods

[0131] 3.1 Reagent Preparation

[0132] Reagent Name Preparation method Storage conditions 3M trifluoroacetic acid 1V trifluoroacetic acid + 3V water Store in a refrigerator at 5℃ Sodium hydride dry powder Wash with 60% sodium hydride and n-hexane Dry at room temperature Sodium borodeuteride sodium hydroxide solution 20mg + 20mM NaOH solution Store in an airtight container 20% acetic acid methanol solution 1V glacial acetic acid + 4V water Store in a refrigerator at 5℃ Polysaccharide methylation kit Anhydrous alkaline solution A Store in a refrigerator at 5℃ Solution B, iodomethane solution

[0133] 3.2 Sample Methylation

[0134] After methylation, hydrolysis, and acetylation, the samples were analyzed by GC-MS and compared with a standard mass spectrum library.

[0135] Weigh 2-3 mg of the polysaccharide sample into a glass reaction flask, add 1 mL of anhydrous DMSO, quickly add methylation reagent A, seal the flask, and dissolve the polysaccharide under sonication. Then add methylation reagent B. React in a magnetically stirred water bath at 30°C for 60 min. Finally, add 2 mL of ultrapure water to the mixture to terminate the methylation reaction.

[0136] Take the methylated polysaccharide, add 1 ml of 2M trifluoroacetic acid (TFA) for hydrolysis for 90 min, and evaporate to dryness using a rotary evaporator. Add 2 ml of double-distilled water to the residues, reduce with 60 mg of sodium borohydride for 8 hours, neutralize with glacial acetic acid, rotary evaporate, dry in a 101°C oven, then add 1 ml of acetic anhydride for acetylation, react at 100°C for 1 h, and cool. Then add 3 mL of toluene, concentrate under reduced pressure and evaporate to dryness, repeat 4-5 times to remove excess acetic anhydride.

[0137] 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 the mixture was shaken thoroughly. The supernatant was removed, and this process was repeated four times. The CH2Cl2 layer was dried with an appropriate amount of anhydrous sodium sulfate, and the volume was adjusted to 10 mL, which was then transferred to a liquid chromatography vial. The acetylated product sample was analyzed using a Shimadzu GCMS-QP 2010 gas chromatography-mass spectrometry system.

[0138] GC-MS conditions: RXI-5SIL MS column 30m*0.25mm*0.25um; temperature program conditions: initial temperature 120℃, increase to 250℃ / min at 3℃ / min; hold for 5min; injection port temperature 250℃, detector temperature 250℃, carrier gas helium, flow rate 1mL / min.

[0139] 4. Experimental Results

[0140] GCMS chromatogram of sample (PMAA), such as Figure 8 As shown.

[0141] Results analysis of polysaccharide methylated sugar alcohol acetyl ester (PMAA) are shown in the table below and as follows. Figure 11-14 As shown.

[0142]

[0143] IV. Nuclear Magnetic Resonance Spectra and Analysis

[0144] 1.1. Experimental Materials and Instruments

[0145] Heavy water (D2O, 99.9%) and deuterated acetone were used as internal controls; freeze dryer and Bruker 600M nuclear magnetic resonance spectrometer were used for filtration.

[0146] 2. Experimental Procedure

[0147] Weigh 50 mg of the polysaccharide sample, dissolve it in 0.5 ml of heavy water, and freeze-dry. Then, dissolve the freeze-dried powder again in 0.5 ml of heavy water and continue freeze-drying. Repeat this process to ensure sufficient exchange of active hydrogens. Next, dissolve the sample in 0.5 ml of heavy water and measure its 1H NMR, 13C NMR, DEPT135 one-dimensional, and two-dimensional spectra at 600 MHz at room temperature (25°C).

[0148] 3. Experimental Results

[0149] The proton NMR signal is mainly concentrated between 3.0 and 5.5 ppm. The signal from protons in the sugar ring is between 3.2 and 4.0 ppm, with the main terminal proton peaks at 4.43, 4.45, 4.47, and 4.66 ppm. The signal peaks are concentrated in the 4.3–5.5 ppm region. Figure 13 As shown.

[0150] Carbon spectroscopy analysis in 13 C NMR (201MHz, D2O): The carbon NMR signal is mainly concentrated between 60-120 ppm. Observing the carbon spectrum, the main anodic carbon signal peaks (δ 103.81, 103.82, 103.88, 104.13) are observed, with the anodic carbon region mainly between δ 93 and 105. The ppm regions are δ 76.68, 74.08, 82.67, 76.62, 70.19, 74.15, 71.56, 80.14, 76.67, 61.66, 74.75, 85.63, 77.17, 76.97, 62.03, 74.76, 74.35, 77.31, 69.61, and 62.04 ppm. Based on the monosaccharide composition, this polysaccharide is composed of glucose, indicating that the polysaccharide is mainly glucan. Figure 14 As shown.

[0151] Spectral analysis of Dept135 showed that the peaks at 70.19, 61.66, 62.03, and 62.04 ppm were inverted peaks, indicating a chemical shift at C6. Figure 15 As shown.

[0152] like Figure 16-19 As shown, the HSQC spectrum reveals an anomeric carbon signal of δ 104.13 and a corresponding anomeric hydrogen signal of δ 4.66. Using HH-COSY, the signals for H1-2 are 4.66 / 3.3; H2-3 is 3.3 / 3.42; and H3-4 is 3.42 / 3.66. We can deduce that H1, H2, H3, and H4 have corresponding δ values ​​of 4.66, 3.3, 3.42, and 3.66, respectively. The corresponding δ values ​​are 104.13, 74.76, 74.35, and 77.31. Using HH-COSY, the signals for H6b-6a are 3.81 / 3.62; and for H6a-5 is 3.62 / 3.46. We can deduce that H6b, H6a, and H5 have corresponding δ values ​​of 3.81, 3.62, and 3.46, respectively. The corresponding C6 and C5 values ​​are δ62.04 and 74.54, respectively. Therefore, this signal should be attributed to the glycosidic bond β-Glcp-(1→).

[0153] The HSQC spectrum shows an anomeric carbon signal of δ 103.81, and the corresponding anomeric hydrogen signal of δ 4.45. Using HH-COSY, the signals for H1-2 are 4.45 / 3.25; for H2-3, 3.25 / 3.42; and for H3-4, 3.42 / 3.64. Therefore, we can deduce that the δ values ​​for H1, H2, H3, and H4 are 4.45, 3.25, 3.42, and 3.42, respectively. The value of 3.64 corresponds to C1-4 values ​​of δ103.81, 76.68, 74.08, and 82.67. Dept135, combined with HSQC, indicates that δ3.78 and 4.13 belong to H6a and b peaks, with a corresponding C6 value of 70.19 ppm. In the HMBC plot, H6b intersects C5 and C4 at 3.78 / 76.62 and 3.78 / 82.67, respectively, thus C5 is 76.62 ppm. The corresponding H5 value via HSQC is 3.82 ppm. Therefore, this signal should be attributed to the glycosidic bond →4,6)-β-D-Glcp-(1→).

[0154] The HSQC spectrum shows an anomeric carbon signal of δ 103.88 and a corresponding anomeric hydrogen signal of δ 4.47. Using HH-COSY, the signals for H1-2 are 4.47 / 3.47, and for H2-3, 3.47 / 3.65. We can deduce that H1, H2, and H3 are δ 4.47, 3.47, and 3.65, respectively, with corresponding C1-3 signals of δ 103.88, 74.75, and 85.63. In the HMBC spectrum, 85.63 / 3.44 and 76.97 / 3.71 are assigned to C3 / H5 and C5 / H4, respectively. Using Dept135 and HSQC, the peaks at δ 3.65 and 3.83 are identified as H6a and b, with a corresponding C6 signal of 62.03 ppm. Therefore, this signal should be attributed to the glycosidic bond →3,4)-β-D-Glcp-(1→).

[0155] The HSQC spectrum shows an anomeric carbon signal of δ 103.82 and a corresponding anomeric hydrogen signal of δ 4.43. HH-COSY analysis reveals the signals for H1-2 to be 4.43 / 3.28 and for H2-3 to be 3.28 / 3.44. We can deduce that H1, H2, and H3 are δ 4.43, 3.28, and 3.44, respectively, corresponding to C1-3 values ​​of δ 103.82, 74.15, and 76.82. C4 shifts to a lower field, and the 80.14 ppm signal is attributed to C4. Dept135 combined with HSQC analysis identifies peaks of δ 3.68 and 3.84 as H6a and b, with a corresponding C6 value of 61.66 ppm. Therefore, this signal should be attributed to the glycosidic bond →4)-β-D-Glcp-(1→).

[0156] Based on similar patterns and combined with HMBC and NOESY, all glycosidic bond signals were assigned as follows: Hydrogen and Carbon Signal Assignments

[0157]

[0158] Main chain analysis:

[0159] In the HMBC spectrum, based on the one-dimensional and two-dimensional NMR spectra, we assigned the glycosidic bond signals of the polysaccharide; the anomeric carbon of the glycosidic bond →4)-β-D-Glcp-(1→ has a related signal peak with its own H4; indicating the existence of the →4)-β-D-Glcp-(1→4)-β-D-Glcp-(1→ linkage.

[0160] The anomeric hydrogen of the glycosidic bond →4)-β-D-Glcp-(1→ has a related signal peak with its C4 of →3,4)-β-D-Glcp-(1→, indicating the existence of a →4)-β-D-Glcp-(1→3,4)-β-D-Glcp-(1→ linkage.

[0161] The anomeric hydrogen of the glycosidic bond →3,4)-β-D-Glcp-(1→ has a related signal peak with its C4 of →4,6)-β-D-Glcp-(1→, indicating the existence of a →3,4)-β-D-Glcp-(1→4,6)-β-D-Glcp-(1→ linkage.

[0162] Branch analysis:

[0163] The anomeric hydrogen of the glycosidic bond β-D-Glcp-(1→) has a related signal peak with its C3 of →3,4)-β-D-Glcp-(1→, indicating the existence of a β-D-Glcp-(1→) linkage.

[0164] The anomeric carbon of the glycosidic bond β-D-Glcp-(1→) has a related signal peak with its →4,6)-β-D-Glcp-(1→ H6, indicating the existence of a β-D-Glcp-(1→) linkage.

[0165] In summary, we can deduce that the main chain of this polysaccharide is a β-1,4 glucan, and that β-D-Glcp-(1→ is connected to the main chain by →3,4)-β-D-Glcp-(1→'s o-3 and →4,6)-β-DGlcp-(1→'s o-6 bonds, as shown in the simplified structure below.

[0166]

[0167] Another objective of this invention is to provide an application of Ganoderma lucidum polysaccharide GLP-1, which 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. In particular, 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.

[0168] The Ganoderma lucidum polysaccharide GLP-1 is used in drugs that inhibit tumor spread or enhance human immunity.

[0169] Experimental data on the antitumor effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on Lewis lung cancer-bearing mice

[0170] Experimental Objective

[0171] We used Lewis lung cancer cells to inoculate the right axilla of C57 mice to prepare tumor-bearing mice, and studied the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-1 on lung cancer tumor-bearing mice, so as to provide experimental evidence for its clinical research.

[0172] Experimental materials

[0173] Test sample

[0174] Ganoderma lucidum polysaccharide GLP-1, batch number: ALM20200518A1; provided by Shenzhen Aolimei Tumor Medical Technology Co., Ltd.

[0175] Positive control

[0176] Kanglaite soft capsules, batch number: 20200802, product of Zhejiang Kanglaite Pharmaceutical Co., Ltd.; Cisplatin injection, batch number: 601210104, product of Jiangsu Hansoh Pharmaceutical Group Co., Ltd.

[0177] Experimental animals

[0178] 80 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 number of experimental animals: 430727211103153225, 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.

[0179] Main reagents

[0180] 0.9% Sodium chloride injection, batch number: 21071401C, product of Hunan Kangyuan Pharmaceutical Co., Ltd.; ALP assay kit, batch number: 201223, purchased from Mike Biological Co., Ltd.; TP assay kit, batch number: 105467, ALB assay kit, batch number: 105468, ALT assay kit, batch number: 105475, AST assay kit, batch number: 104446, TBIL assay kit, batch number: 105465, GGT assay kit, batch number: 104447, all products of Wako Pure Chemical Industries, Ltd.

[0181] Main instruments

[0182] S10 portable high-speed disperser, Shanghai Xinzhi; AR223CN electronic balance, Ohaus Instruments (Changzhou) Co., Ltd.; LABOSPECT003 automated biochemical analyzer, Hitachi, Japan; TDZ5-WS benchtop multi-tube automatic balancing centrifuge, Hunan Kaida Industrial Development Co., Ltd.; ME2002E electronic balance, Mettler Toledo Instruments (Shanghai) Co., Ltd.; digital display vernier calipers, Yongkang Zhengfeng Hardware Co., Ltd.; flow cytometer, BD Corporation; ASP200S fully automatic tissue dehydrator, ASP300S fully automatic tissue dehydrator, TP1020 fully automatic dehydrator, HI1210 slide spreader, HI1220 slide roaster, RM2235 paraffin sectioner, EG1150H+C tissue embedding machine, AutoStainer. XL Automatic Slide Staining Machine + CV5030 Automatic Cover Slider, Leica GmbH, Germany; BX43 Biological Microscope + MD50 Digital Imaging System, CX31 Biological Microscope, Olympus Corporation, Japan.

[0183] Experimental methods

[0184] Take 1×10 Lewis tumor cell lines in logarithmic growth phase 7 0.2 mL of the drug was injected into the right axilla of eight normal male C57 mice. Tumor growth was observed to prepare tumor-bearing mice. Tumors were cultured until the tumor volume reached 200 mm². 3 At the above time, tumor tissue from tumor-bearing mice was aseptically removed and a tissue cell suspension was prepared. The suspension was then homogenized with cell culture medium at a 1:1 (V:V) ratio and injected into the right axilla of 62 male C57 mice at a volume of 0.2 mL per mouse. The injection was continued until the tumor volume reached 100 mm². 3 In the above-mentioned cases, well-grown tumor-bearing mice without ulceration were selected and randomly divided into four groups according to tumor volume: model control group, cisplatin group (4 mg / kg), Kanglaite soft capsule group (1404 mg / kg), cisplatin + Kanglaite soft capsule group (4 + 1404 mg / kg), cisplatin + GLP-1 low-dose group (4 + 130 mg / kg), and cisplatin + GLP-1 high-dose group (4 + 1170 mg / kg), with 8 mice in each group. An additional 8 mice served as a normal control group. In the cisplatin group and the combination groups, cisplatin was administered once every 3 days, Kanglaite once daily, and GLP-1 once daily, with administration volumes of 20 mL / kg (gavage) and 10 mL / kg (intraperitoneal injection), respectively, for 27 consecutive days. After the last administration, blood samples were collected from the orbital cavity to detect blood WBC, RBC, liver and kidney function (ALT, AST, BUN, CRE), and CD4+ and CD8+ levels. The spleen, thymus, and tumor were weighed and the organ-to-body ratio was calculated. Bone marrow samples were collected for smear examination, and the tumor, spleen, stomach, and kidneys were subjected to histopathological analysis.

[0185] Dosage design

[0186] Based on previous research results, Ganoderma lucidum polysaccharide GLP-1 was designed with a low dose of 130 mg / kg and a high dose of 1170 mg / kg. The corresponding drugs were administered according to Table 1. This trial was conducted at a multiple of the clinically intended dose.

[0187] The proposed clinical dosage of Kanglaite soft capsules is 0.45g / capsule, 6 capsules / dose, 4 times / day, which is 10.8g / day. Converted to the equivalent dose for mice based on body surface area, this is 10.8g / day * 0.0026 / 0.02kg = 1404mg / kg. This trial will be conducted at a multiple of the proposed clinical dosage.

[0188] The cisplatin dosage design was based on the clinical dosage of cisplatin not exceeding 100 mg / m2 per person per day and the tolerance of mice to cisplatin, and a dosage of 4 mg / kg was selected as the dosing dose.

[0189] Table 1. Trial grouping and dosage design

[0190]

[0191] detection indicators

[0192] therapeutic indicators

[0193] Evaluation of animal quality of life and survival time: During the experiment, detailed records were kept of the weight of each group of animals, animal mortality, mental state of mice, fur color, fur smoothness and stool form, and whether there was diarrhea.

[0194] Tumor volume measurement: The length and width of the mouse tumor were measured with calipers every 3 days after drug administration, and the tumor volume was calculated. Tumor volume (TV) = 1 / 2 × a (length) × b 2 (Width). Relative tumor volume (RTV) = TV t / TV0, where TV0 is the tumor volume at the time of drug administration, TV t This represents the tumor volume measured each time. Relative tumor proliferation rate T / C (%) = T RTV / C RTV ×100, T RTV Treatment group RTV; C RTV Model control group RTV. Tumor growth inhibition rate (%) = (tumor weight of model control group - tumor weight of treatment group) / tumor weight of model control group × 100%. A tumor growth inhibition rate (%) < 40% is considered ineffective; a tumor growth inhibition rate (%) ≥ 40% and P ≤ 0.05 is considered effective.

[0195] Spleen and thymus organ coefficients: After the last administration, the spleen, thymus, and tumor were weighed and the organ coefficients were calculated. Organ coefficient (%) = organ weight / body weight after fasting × 100%.

[0196] Reduce toxicity and enhance efficacy

[0197] Hematological tests: Blood samples were collected from the orbital rim after the last administration to perform complete blood count (WBC, RBC) and liver and kidney function tests (BUN, CRE).

[0198] Bone marrow examination: Bone marrow was collected after the last administration and smears were prepared to observe changes in bone marrow cells in each group of mice.

[0199] Histopathological examination: After the last administration, tumor tissue, spleen, stomach, and kidney were taken for HE staining and histopathological changes were observed.

[0200] Blood CD4+ and CD8+ levels were measured by flow cytometry after the last administration of the drug.

[0201] Data processing and statistical analysis

[0202] Data from this experiment were rounded to the nearest whole number and statistically analyzed according to the center's standard operating procedures (SOPs). SPSS software was used for statistical analysis. Quantitative data were expressed as mean ± standard deviation (x ± s). Normality and homogeneity of variance were tested using Leven's test. If there was no statistical significance (P > 0.05), one-way ANOVA was used. 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), Dunnett's test (non-parametric method) was used for comparative analysis. The statistical results were compared with α = 0.05, where P ≤ 0.05 indicated statistical significance, and P ≤ 0.01 indicated a highly significant difference.

[0203] Experimental results

[0204] General clinical observation and animal mortality

[0205] Before administration, the mice were active and their movement and gait were normal. The model control group 2M06 and 2M03 died on January 20, 2022 and January 21, 2022, respectively; the Kanglaite soft capsule group 4M01 died on January 19, 2022; the cisplatin + Kanglaite soft capsule group 5M01 and 5M04 died on January 14, 2022 and January 22, 2022, respectively; and the cisplatin + GLP-1 high-dose group 7M08 died on January 9, 2022.

[0206] The mortality rates for each group were 0%, 25.0%, 0%, 12.5%, 25.0%, 0%, and 12.5%, respectively.

[0207] Table 2. Statistics on the number of surviving animals and mortality rate in each group.

[0208]

[0209] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on body weight in Lewis lung cancer-bearing mice

[0210] As shown in Table 3, compared with the normal control group, the body weight of mice in the model control group was significantly reduced on day 4 after administration (P≤0.05). Compared with the model control group, the body weight of mice in the cisplatin group and the cisplatin combined with Kanglaite soft capsule group was significantly reduced on days 7–25 after administration (P≤0.01); the body weight of mice in the cisplatin combined with low and high dose GLP-1 groups was significantly reduced on days 4–25 after administration (P≤0.05 or P≤0.01); and the body weight of mice in the Kanglaite soft capsule group was significantly reduced on days 10–19 after administration.

[0211] Compared with the cisplatin group, mice in the Kanglaite soft capsule group showed a significant increase in body weight on days 7-25 after administration (P≤0.05 or P≤0.01), while mice in the cisplatin combined with high-dose GLP-1 group showed a significant decrease in body weight on day 7 after administration (P≤0.05). Compared with the Kanglaite soft capsule group, mice in the cisplatin combined with Kanglaite soft capsule group, as well as the low- and high-dose GLP-1 groups, showed a significant decrease in body weight on days 7-25 after administration (P≤0.05 or P≤0.01). There were no statistically significant differences among the groups compared with the cisplatin + Kanglaite soft capsule group.

[0212] Table 3. Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on body weight in Lewis cell tumor-bearing mice.

[0213]

[0214]

[0215] Note: Compared with the normal control group, + P≤0.05; Compared with the model control group, *P≤0.05, **P≤0.01; Compared with the cisplatin group, #P≤0.05, ## P≤0.01; compared with the Kanglaite soft capsule group, & P≤0.05, && P≤0.01, compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0216] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on tumor volume and relative tumor volume in Lewis lung cancer-bearing mice

[0217] As shown in Table 4, compared with the model control group, the tumor volume of mice in the cisplatin combined with low- and high-dose GLP-1 groups was significantly reduced on days 10–25 (P≤0.05 or P≤0.01), the tumor volume of mice in the cisplatin group was significantly reduced on days 10 and 16–25 (P≤0.05 or P≤0.01), and the tumor volume of mice in the cisplatin combined with Kanglaite soft capsule group was significantly reduced on days 13–25 (P≤0.05 or P≤0.01); there was no significant difference in tumor volume in the Kanglaite soft capsule group. Compared with the cisplatin group, the tumor volume of mice in the cisplatin combined with high-dose GLP-1 group was significantly reduced on days 22–25 (P≤0.05), and the tumor volume of mice in the Kanglaite soft capsule group was significantly increased on days 7–25 (P≤0.05 or P≤0.01). Compared with the Kanglaite soft capsule group, the tumor volume of mice in the cisplatin combined with GLP-1 low-dose group, high-dose group, and Kanglaite soft capsule group was significantly reduced on days 13 to 25 (P≤0.05 or P≤0.01).

[0218] As shown in Table 5, compared with the model control group, the relative tumor volume of mice in the cisplatin group, the cisplatin combined with Kanglaite soft capsule group, and the cisplatin combined with low and high dose GLP-1 groups was significantly reduced on days 13–25 (P≤0.05 or P≤0.01). There was no statistically significant difference in relative tumor volume in the Kanglaite soft capsule group. Compared with the cisplatin + Kanglaite soft capsule group, the tumor volume of mice in the cisplatin combined with high dose GLP-1 group was significantly reduced on days 19 and 25 (P≤0.05), while there were no statistically significant differences in the other groups.

[0219] Table 4. Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on tumor volume in Lewis lung cancer-bearing mice.

[0220]

[0221] Note: Compared with the model control group, *P≤0.05, **P≤0.01; compared with the cisplatin group, # P≤0.05, ## P≤0.01; compared with the Kanglaite soft capsule group, & P≤0.05,&& P≤0.01, compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0222] Table 5. Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on relative tumor volume in Lewis cell tumor-bearing mice (x±s)

[0223]

[0224] Note: Compared with the model control group, *P≤0.05, **P≤0.01.

[0225] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on relative tumor proliferation rate (T / C) in Lewis lung cancer-bearing mice

[0226] As shown in Table 6, the T / C values ​​of mice in the low-dose cisplatin + GLP-1 group from D7 to D25 were 50.0%, 21.2%, 3.5%, 3.3%, 7.2%, 11.0%, and 11.3%, respectively, all ≤60.0%; the T / C values ​​of mice in the high-dose cisplatin + GLP-1 group from D10 to D25 were 27.3%, 0.0%, 0.6%, 0.4%, 1.4%, and 0.8%, respectively, all ≤60.0%. All values ​​were ≤60.0%; in the cisplatin group, the T / C rates for D7–D10 and D16–D25 were 45.5%, 27.3%, 28.2%, 36.9%, 30.9%, and 23.6%, respectively, all ≤60.0%; in the cisplatin + Kanglaite soft capsule group, the T / C rates for D13–D25 were 7.3%, 3.3%, 15.6%, 7.3%, and 15.6%, respectively, all ≤60.0%.

[0227] Table 6. Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on relative tumor proliferation rate in Lewis lung cancer-bearing mice.

[0228]

[0229] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on organ coefficient and tumor growth inhibition rate in Lewis lung cancer-bearing mice

[0230] As shown in Table 7, compared with the normal control group, the spleen and tumor 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, thymus, and tumor coefficients of mice in the cisplatin group, the cisplatin combined with GLP-1 low-dose group, and the Kanglaite soft capsule group were significantly decreased (P≤0.05 or P≤0.01), and the spleen and tumor coefficients of mice in the cisplatin combined with GLP-1 high-dose group were significantly decreased (P≤0.01). Compared with the cisplatin group, the tumor coefficient of mice in the cisplatin combined with GLP-1 high-dose group was significantly decreased (P≤0.05), while the spleen and tumor coefficients of mice in the Kanglaite soft capsule group were significantly increased (P≤0.01). Compared with the Kanglaite soft capsule group, the spleen and tumor coefficients of mice in the cisplatin combined with GLP-1 low-dose and high-dose groups were significantly decreased (P≤0.01). Compared with the cisplatin + Kanglaite soft capsule group, there was no statistically significant difference in the organ-to-body ratio among the groups. The tumor growth inhibition rate in the Kanglaite soft capsule group was -6.8%, with a value <40.0%; the tumor growth inhibition rates in the cisplatin group, the cisplatin combined with Kanglaite soft capsule group, and the low- and high-dose GLP-1 groups were 61.6%, 78.3%, 77.6%, and 96.7%, respectively, with values ​​≥40.0%.

[0231] Table 7. Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on organ coefficient and tumor growth inhibition rate in Lewis lung cancer-bearing mice.

[0232]

[0233] Note: Compared with the normal control group, ++ P≤0.01; Compared with the model control group, *P≤0.05, **P≤0.01; Compared with the cisplatin group, # P≤0.05, ## P≤0.01; compared with the Kanglaite soft capsule group, & P≤0.05, && P≤0.01; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0234] exist Figure 20 Control groups are labeled as follows: 1: Model control group; 2: Cisplatin group; 3: Kanglaite soft capsule group; 4: Cisplatin + Kanglaite soft capsule group; 5: Cisplatin + GLP-1 low-dose group; 6: Cisplatin + GLP-1 high-dose group.

[0235] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on blood biochemical parameters in Lewis lung cancer-bearing mice

[0236] As shown in Table 8, compared with the normal control group, the levels of blood WBC, ALT, AST, and BUN in the model control group mice were significantly increased (P≤0.05 or P≤0.01), while RBC was significantly decreased (P≤0.05). Compared with the model control group, the levels of blood WBC and AST in the cisplatin combined with high-dose GLP-1 group were significantly decreased (P≤0.05 or P≤0.01), the levels of blood WBC, ALT, and AST in the cisplatin combined with low-dose GLP-1 group were significantly decreased (P≤0.05), and the WBC levels in both the cisplatin group and the cisplatin combined with Kanglaite soft capsule group were significantly decreased (P≤0.05). Compared with the cisplatin group, the levels of blood RBC in the cisplatin combined with high-dose GLP-1 group were significantly increased (P≤0.05). Compared with the Kanglaite soft capsule group, the levels of blood WBC in the cisplatin combined with high-dose GLP-1 group were significantly decreased (P≤0.05), and the levels of blood WBC in the cisplatin combined with Kanglaite soft capsule group were significantly decreased (P≤0.05). Compared with the cisplatin + Kanglaite soft capsule group, the blood WBC count of mice in the cisplatin combined with low-dose GLP-1 group was significantly increased (P≤0.05), while there was no statistically significant difference in the other groups.

[0237] Table 8 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on blood biochemical parameters in Lewis lung cancer-bearing mice

[0238]

[0239] Note: Compared with the normal control group, + P≤0.05, ++ P≤0.01; Compared with the model control group, *P≤0.05, **P≤0.01; Compared with the cisplatin group, # P≤0.05, ## P≤0.01; compared with the Kanglaite soft capsule group, & P≤0.05; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05.

[0240] Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on CD3 in Lewis lung cancer-bearing mice + / CD4 + CD3 + / CD8 + Impact

[0241] As shown in Table 9, compared with the normal control group, the blood CD3 of mice in the model control group was... + / CD8 + Significantly increased (P≤0.05); no statistically significant difference was found between the drug-treated groups and the model control group. Compared with the cisplatin group, the CD3 levels in the blood of mice in the Kanglaite soft capsule group were significantly higher (P≤0.05); + / CD8 +Significantly reduced (P≤0.05). Compared with the Kanglaite soft capsule group, the cisplatin combined with Kanglaite soft capsule group showed significantly lower CD3 levels in the blood of mice. + / CD8 + Significantly increased (P≤0.05). There was no statistically significant difference between the treatment groups and the cisplatin + Kanglaite soft capsule group.

[0242] Table 9. Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on CD3 in Lewis lung cancer-bearing mice. + / CD4 + CD3 + / CD8 + Effect of content

[0243]

[0244] Note: Compared with the normal control group, + P≤0.05; compared with the model control group, *P≤0.05; compared with the cisplatin group, # P≤0.05; compared with the Kanglaite soft capsule group, & P≤0.05, && P≤0.01; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0245] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on bone marrow cells of Lewis lung cancer-bearing mice

[0246] As shown in Table 10, compared with the normal control group, the mononuclear cell system in the bone marrow of mice in the model control group was significantly increased (P≤0.05), while the granulocyte-erythrocyte system, granulocyte-erythrocyte ratio, and other cell types showed an increasing trend, and the erythrocyte system and lymphocyte system showed a decreasing trend, but these changes were not statistically significant. Compared with the model control group, the erythrocyte system in the bone marrow of mice in the cisplatin combined with GLP-1 low-dose group and the Kanglaite soft capsule group was significantly increased (P≤0.05), while the granulocyte-erythrocyte ratio and mononuclear cell system were significantly decreased (P≤0.05). The mononuclear cell system in the bone marrow of mice in the Kanglaite soft capsule group and the cisplatin combined with GLP-1 high-dose group was significantly decreased (P≤0.01). There were no statistically significant differences among the treatment groups compared with the cisplatin group. Compared with the Kanglaite soft capsule group, the bone marrow lymphocyte system in mice in the cisplatin combined with GLP-1 low-dose and high-dose groups was significantly increased (P≤0.05). Compared with the cisplatin + Kanglaite soft capsule group, the cisplatin combined with high-dose GLP-1 group showed a significant increase in the myeloglobin system in the bone marrow (P≤0.05) and a significant decrease in the erythrocyte system (P≤0.05), while the other groups showed no statistical significance.

[0247] Table 10 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on bone marrow cells in Lewis lung cancer-bearing mice

[0248]

[0249] Note: Compared with the normal control group, + P≤0.05; Compared with the model control group, *P≤0.05, **P≤0.01; Compared with the cisplatin group, # P≤0.05, ## P≤0.01; compared with the Kanglaite soft capsule group, & P≤0.05, && P≤0.01; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0250] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the pathological characteristics of tumors, spleen, kidneys, and stomach tissues in Lewis cell tumor-bearing mice.

[0251] like Figure 21 As shown, in the model control group, the number of tumor cells around the tumor was abundant, the cells were densely packed, the cell volume was large, the nuclear-cytoplasmic ratio was high, and mitotic figures were frequently observed. In the cisplatin group, large areas of necrosis and hemorrhage were visible in the center of the tumor, and the number of tumor cells was abundant. In the cisplatin + Kanglaite soft capsule group and the cisplatin + high-dose GLP-1 group, necrosis was visible in the center of the tumor tissue, and the number of surrounding tumor cells was reduced.

[0252] like Figure 22 , 23 As shown, no obvious abnormalities were found in the pathological examination of the kidneys and stomach in each group.

[0253] like Figure 24 As shown, the spleen in the model control group showed significant red pulp hematopoiesis with diverse cell types. No significant abnormalities were observed in the spleen of the cisplatin + Kanglaite soft capsule group, while the spleens of the other groups showed varying degrees of red pulp hematopoiesis with diverse cell types.

[0254] exist Figure 21 Control groups are labeled as follows: A: Normal control group; B: Model control group; C: Cisplatin group; D: Kanglaite soft capsule group; E: Cisplatin + Kanglaite soft capsule group; F: Cisplatin + GLP-1 low-dose group; G: Cisplatin + GLP-1 high-dose group.

[0255] exist Figure 22 , 23 Control groups are labeled as follows: A: Normal control group; B: Model control group; C: Cisplatin group; D: Kanglaite soft capsule group; E: Cisplatin + Kanglaite soft capsule group; F: Cisplatin + GLP-1 low-dose group; G: Cisplatin + GLP-1 high-dose group.

[0256] exist Figure 24Control groups are labeled as follows: A: Normal control group; B: Model control group; C: Cisplatin group; D: Kanglaite soft capsule group; E: Cisplatin + Kanglaite soft capsule group; F: Cisplatin + GLP-1 low-dose group; G: Cisplatin + GLP-1 high-dose group.

[0257] in conclusion

[0258] Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin can significantly inhibit the growth of tumors in Lewis lung cancer-bearing mice and has a significant synergistic effect.

[0259] Discussion and Summary

[0260] In recent years, with the gradual deterioration of the environment and factors such as smoking, the incidence of lung cancer has been rising year by year, with a high mortality rate and a trend towards affecting younger people. Clinically, the main treatment methods are surgery, radiotherapy, and chemotherapy to achieve early tumor eradication and reduce tumor burden. However, due to the highly invasive, metastatic, and insensitive nature of lung cancer cells to radiotherapy and chemotherapy, patients often experience immunosuppression after surgery, which accelerates tumor metastasis and development, leading to poor clinical prognosis.

[0261] The results of this experiment showed that the tumor volume of mice in the model control group was significantly increased, the thymus index was significantly decreased, the spleen index was significantly increased, the number of red blood cells in the blood was significantly increased, the number of white blood cells was significantly decreased, and liver and kidney function was significantly abnormal. In addition, CD3 + The content increased significantly, CD4 + Increased content, CD8 + The decrease indicates that the lymphatic system function was reduced in the model control mice during tumor development.

[0262] Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin significantly inhibited tumor growth in mice, significantly reduced thymus and spleen indices, significantly decreased red blood cell count and significantly increased white blood cell count in mouse blood, and gradually restored liver and kidney function indicators to normal levels; CD4+ in mouse blood... + The content decreased, CD8 + The increased content indicates that the combined use of GLP-1 and cisplatin can protect immune organs and enhance the body's own immune function, thereby reducing toxicity and improving anti-tumor effects. Compared with the Kanglaite soft capsule group and the cisplatin combined with Kanglaite soft capsule group, Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin can significantly inhibit tumor growth, and the tumor growth inhibition effect of Ganoderma lucidum polysaccharide GLP-1 is significantly stronger than that of Kanglaite soft capsule.

[0263] In conclusion, Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin can significantly inhibit the growth of Lewis lung cancer tumors in mice and has a significant synergistic effect.

[0264] Experimental data on the antitumor effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on Lewis lung cancer-bearing mice

[0265] Research purpose

[0266] In this experiment, Lewis lung cancer cells were inoculated under the right axilla of C57 mice to prepare tumor-bearing mice, and the mechanism of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy to increase chemotherapy tolerance was studied, providing an experimental basis for its clinical research.

[0267] Experimental materials

[0268] Test samples

[0269] Ganoderma lucidum polysaccharide GLP-1, batch number: ALM20200518A1; provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0270] Positive control

[0271] Kanglaite soft capsules, batch number: 20210904, Zhejiang Kanglaite Pharmaceutical Co., Ltd.; Cisplatin injection, batch number: 601211, Jiangsu Hansoh Pharmaceutical Group Co., Ltd.

[0272] Experimental animals

[0273] 95 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(Hunan)2019 - 0004, quality certificate number of experimental animals: 430727211103153225, raised in area D of the barrier environment animal laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd., use license number of experimental animals: SYXK(Hunan)2020 - 0015.

[0274] Main reagents

[0275] 0.9% sodium chloride injection, batch number: 21071401C, product of Hunan Kangyuan Pharmaceutical Co., Ltd.; ALT assay kit, batch number: 201751, AST assay kit, batch number: 110620, CRE assay kit, batch number: 111644, BUN assay kit, batch number: 201749, all products of Wako Pure Chemical Industries, Ltd.

[0276] [[ID=3​​S10 portable high-speed disperser, Shanghai Xinzhi; AR223CN electronic balance, Ohaus Instruments (Changzhou) Co., Ltd.; LABOSPECT003 automated biochemical analyzer, Hitachi, Japan; TDZ5-WS benchtop multi-tube automatic balancing centrifuge, Hunan Kaida Industrial Development Co., Ltd.; ME2002E electronic balance, Mettler Toledo Instruments (Shanghai) Co., Ltd.; digital display vernier calipers, Yongkang Zhengfeng Hardware Co., Ltd.; flow cytometer, BD Corporation; ASP200S fully automatic tissue dehydrator, ASP300S fully automatic tissue dehydrator, TP1020 fully automatic dehydrator, HI1210 slide spreader, HI1220 slide roaster, RM2235 paraffin sectioner, EG1150H+C tissue embedding machine, AutoStainer. XL Automatic Slide Staining Machine + CV5030 Automatic Cover Slider, Leica GmbH, Germany; BX43 Biological Microscope + MD50 Digital Imaging System, CX31 Biological Microscope, Olympus Corporation, Japan.

[0278] Research Content

[0279] Experimental methods

[0280] Take 1×10 Lewis tumor cell lines in logarithmic growth phase 7 0.2 mL of the drug was injected into the right axilla of 10 normal male C57 mice. Tumor growth was observed to prepare tumor-bearing mice. Tumors were cultured until the tumor volume reached 200 mm². 3 At the above time, tumor tissue from tumor-bearing mice was aseptically removed and a tissue cell suspension was prepared. The suspension was then homogenized with cell culture medium at a 1:1 (V:V) ratio and injected into the right axilla of 72 male C57 mice at a volume of 0.2 mL per mouse. The injection was continued until the tumor volume reached 100 mm². 3In the above-mentioned cases, well-grown tumor-bearing mice without ulceration were selected and randomly divided into four groups according to tumor volume: model control group, radiotherapy group (2 Gy / d), Kanglaite soft capsule group (1404 mg / kg), radiotherapy + Kanglaite soft capsule group (2 Gy + 1404 mg / kg), radiotherapy + low-dose GLP-1 group (2 Gy + 130 mg / kg), and radiotherapy + high-dose GLP-1 group (2 Gy + 1170 mg / kg), with 10 mice in each group. Ten mice were also used as a normal control group. Except for the normal control group, the remaining animals underwent radiotherapy using an animal radiotherapy device. The animals were anesthetized, placed in a self-made lead apron, and exposed to radiotherapy with a radiation intensity of 2 Gy / day for 5 consecutive days. Mice in the normal control group, model control group, and radiotherapy group were administered distilled water by gavage, while mice in the chemotherapy group were injected intraperitoneally with cisplatin. The remaining groups were administered the corresponding drug solutions. The dosage was 20 mL / kg (by gavage), once daily for 14 consecutive days. After the last administration, blood samples were collected from the orbital rim to measure the levels of white blood cells (WBC), red blood cells (RBC), and liver and kidney function indicators (ALT, AST, CRE, BUN). CD3+ levels in the blood were also measured by flow cytometry. + CD4 + CD8 + Content; after weighing the spleen, thymus, and tumor, the organ-to-body ratio was calculated, and the spleen, thymus, stomach, kidney, and nose were taken for histopathological examination.

[0281] Dosage design

[0282] Based on previous research results, Ganoderma lucidum polysaccharide GLP-1 was designed with a low dose of 130 mg / kg and a high dose of 1170 mg / kg. The corresponding drugs were administered according to Table 11. This trial was conducted at a multiple of the clinically intended dose.

[0283] The proposed clinical dosage of Kanglaite soft capsules is 0.45g / capsule, 6 capsules / dose, 4 times / day, which is 10.8g / day. Converted to the equivalent dose for mice based on body surface area, this is 10.8g / day * 0.0026 / 0.02kg = 1404mg / kg. This trial will be conducted at a multiple of the proposed clinical dosage.

[0284] This trial was designed with a radiotherapy intensity of 2 Gy / day, which is an equal multiple of the clinically intended dose.

[0285] Table 11 Trial grouping and dosage design

[0286]

[0287]

[0288] detection indicators

[0289] therapeutic indicators

[0290] Evaluation of animal quality of life and survival time: During the experiment, detailed records were made of the weight of each group of animals, the mortality of animals, the mental state of mice, fur color, fur smoothness and stool form, and whether there was diarrhea, etc.

[0291] Tumor volume measurement: The length and width of the mouse tumor were measured with calipers every 3 days after drug administration, and the tumor volume was calculated. Tumor volume (TV) = 1 / 2 × a (length) × b 2 (Width). Relative tumor volume (RTV) = TV t / TV0, where TV0 is the tumor volume at the time of drug administration, TV t This represents the tumor volume measured each time. Relative tumor proliferation rate T / C (%) = T RTV / C RTV ×100%, T RTV Treatment group RTV; C RTV : Model control group RTV.

[0292] Immune organs: After the last administration, the spleen and thymus were harvested and weighed, and the organ coefficient was calculated. All animal indicators were photographed and recorded. Organ coefficient (%) = organ weight / body weight after fasting × 100%.

[0293] Hematological tests: Complete blood count (WBC, RBC) and biochemical (liver and kidney function) indicators after the last administration.

[0294] Histopathological examination: After the last administration, the thymus, spleen, nasal tissue, stomach, and kidney were taken for HE staining, sectioning, and embedding for pathological examination.

[0295] CD4 + CD8 + Content detection: After the last administration, blood lymphocyte typing CD3 was performed using flow cytometry. + / CD4 + CD3 + / CD8 + content.

[0296] Data processing and statistical analysis

[0297] The data from this experiment were rounded to the nearest whole number and statistically analyzed according to the center's standard operating procedures (SOPs). SPSS was used for statistical analysis. Quantitative data are expressed as mean ± standard deviation. The results indicate that Leven's test was used to test for normality and homogeneity of variance. If there was no statistical significance (P>0.05), one-way 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), Dunnett's test (nonparametric method) was used for comparative analysis. The statistical results were presented with α = 0.05 as the test limit, where P≤0.05 indicated statistical significance, and P≤0.01 indicated a highly significant difference.

[0298] Experimental results

[0299] General clinical observation and animal mortality

[0300] Before administration, the mice were active and their movements and gait were normal; after administration, the weight of the radiotreated mice decreased. Mice in the radiotherapy group 3M01, 3M10, 3M03, and 3M02 died on days 10, 11, and 12, respectively; mice in the radiotherapy combined with Kanglaite soft capsule group 6M01 and 6M10 died on day 10; mice in the radiotherapy combined with low-dose GLP-1 group 7M05 died on day 7; and mice in the radiotherapy combined with high-dose GLP-1 group 8M08 and 8M05 died on days 7 and 10, respectively.

[0301] The mortality rates for each group were 0%, 0%, 40%, 0%, 20%, 10%, and 20%, respectively.

[0302] Table 12 Statistics on the number of surviving animals and mortality rate in each group

[0303]

[0304]

[0305] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on body weight of Lewis cell tumor-bearing mice

[0306] As shown in Table 13, compared with the normal control group, the body weight of mice in the model control group was significantly reduced on day 1 after administration (P≤0.01). Compared with the model control group, the body weight of mice in the radiotherapy combined with low- and high-dose GLP-1 groups and the Kanglaite soft capsule group was significantly reduced on day 4 after administration; the body weight of mice in the radiotherapy combined with low- and high-dose GLP-1 groups and the Kanglaite soft capsule group was significantly reduced on day 7 after administration (P≤0.01); the body weight of mice in the radiotherapy combined with low- and high-dose GLP-1 groups and the Kanglaite soft capsule group was significantly reduced on day 10 after administration (P≤0.05 or P≤0.01); and the body weight of mice in the radiotherapy combined with low- and high-dose GLP-1 groups and the Kanglaite soft capsule group was significantly reduced on day 13 after administration (P≤0.05 or P≤0.01). Compared with the radiotherapy group, the body weight of mice in the radiotherapy combined with low-dose GLP-1 and high-dose groups, and the Kanglaite soft capsule group was significantly reduced on day 4 (P≤0.05 or P≤0.01); the body weight of mice in the Kanglaite soft capsule group was significantly increased on days 7–10 (P≤0.05 or P≤0.01); the body weight of mice in the radiotherapy combined with Kanglaite soft capsule group was significantly reduced on days 10–13 (P≤0.05); and the body weight of mice in the radiotherapy combined with low-dose GLP-1 group was significantly reduced on day 13 (P≤0.05 or P≤0.01). There were no statistically significant differences between the radiotherapy and Kanglaite soft capsule groups.

[0307] Table 13 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on body weight in Lewis cell tumor-bearing mice (Mean ± SEM)

[0308]

[0309] 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 Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01; compared with the radiotherapy + Kanglaite soft capsule group, ■ P≤0.05, ■■ P≤0.01

[0310] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on tumor volume and relative tumor volume in Lewis cell tumor-bearing mice

[0311] As shown in Table 14, compared with the model control group, the tumor volume of mice in the radiotherapy combined with GLP-1 low-dose group and the Kanglaite soft capsule group was significantly reduced on days 7-13 after administration (P≤0.05 or P≤0.01); the tumor volume of mice in the radiotherapy combined with GLP-1 high-dose group was significantly reduced on day 13 after administration (P≤0.05 or P≤0.01); and the tumor volume of mice in the radiotherapy group was significantly reduced on days 7 and 13 after administration (P≤0.01). Compared with the radiotherapy group, the tumor volume of mice in the Kanglaite soft capsule group was significantly increased on day 10 after administration (P≤0.01). Compared with the Kanglaite soft capsule group, the tumor volume of mice in the radiotherapy combined with GLP-1 low-dose group and the Kanglaite soft capsule group was significantly reduced on days 7-13 after administration (P≤0.05 or P≤0.01); and the tumor volume of mice in the radiotherapy combined with GLP-1 high-dose group was significantly reduced on day 7 after administration (P≤0.05).

[0312] As shown in Table 15, compared with the model control group, the relative tumor volume of mice in the radiotherapy combined with Kanglaite soft capsule group and the low-dose GLP-1 group was significantly reduced on days 7-13 after administration (P≤0.05 or P≤0.01); the relative tumor volume of mice in the radiotherapy group was significantly reduced on day 7 after administration (P≤0.05). Compared with the Kanglaite soft capsule group, the relative tumor volume of mice in the radiotherapy combined with GLP-1 low-dose group and the Kanglaite soft capsule group was significantly reduced on days 7-13 after administration (P≤0.05 or P≤0.01).

[0313] Table 14. Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on tumor volume in Lewis cell tumor-bearing mice (Mean ± SEM)

[0314]

[0315]

[0316] Note: 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 Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0317] Table 15 Effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on relative tumor volume in Lewis cell tumor-bearing mice (Mean±SEM)

[0318]

[0319] Note: 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 Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0320] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on relative tumor proliferation rate (T / C) in Lewis cell tumor-bearing mice

[0321] As shown in Table 16, the T / C values ​​of mice in the radiotherapy combined with Kanglaite soft capsule group on days 7-13 after administration were 47.0%, 9.0%, and 13.2%, respectively, all ≤60.0%; the T / C values ​​of mice in the radiotherapy combined with low-dose GLP-1 group on days 7-13 after administration were 22.1%, 25.3%, and 18.3%, respectively, all ≤60.0%.

[0322] Table 16 Effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on relative tumor proliferation rate in mice (Mean±SEM)

[0323]

[0324] Note: Relative tumor proliferation rate: T / C (%) > 60 indicates ineffectiveness; T / C (%) ≤ 60 indicates effectiveness.

[0325] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on organ coefficient and tumor growth inhibition rate in Lewis cell tumor-bearing mice

[0326] As shown in Table 17, compared with the normal control group, the spleen-to-tumor ratio was significantly increased in the model control group (P≤0.01), while the thymus-to-tumor ratio was significantly decreased (P≤0.05). Compared with the model control group, the spleen coefficient was significantly decreased in the radiotherapy group (P≤0.01); the spleen coefficient was significantly decreased in the radiotherapy combined with high-dose GLP-1 group (P≤0.05); the spleen coefficient and tumor coefficient were significantly decreased in the radiotherapy combined with low-dose GLP-1 group (P≤0.05 or P≤0.01); and the thymus coefficient and spleen coefficient were significantly decreased in the radiotherapy combined with Kanglaite soft capsule group (P≤0.01). Compared with the radiotherapy group, the thymus index was significantly increased in the Kanglaite soft capsule group (P≤0.01). Compared with the Kanglaite soft capsule group, the spleen coefficient and tumor coefficient were significantly decreased in the radiotherapy combined with low-dose GLP-1 group (P≤0.01). There were no statistically significant differences among the other groups. The tumor growth inhibition rates for each group were 25.0%, -35.9%, 67.6%, 75.5%, and -8.6%, respectively.

[0327] Table 17 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on organ coefficients and tumor growth inhibition rates in Lewis cell tumor-bearing mice (Mean ± SEM)

[0328]

[0329] 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 Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01; compared with the radiotherapy + Kanglaite soft capsule group, ■ P≤0.05, ■■ P≤0.01.

[0330] like Figure 25 As shown, the corresponding labels are: 2: Model control group; 3: Radiotherapy group; 5: Kanglaite soft capsule group; 6: Radiotherapy + Kanglaite soft capsule group; 7: Radiotherapy + GLP-1 low-dose group; 8: Radiotherapy + GLP-1 high-dose group.

[0331] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on blood biochemical parameters in Lewis cell tumor-bearing mice

[0332] As shown in Table 18, compared with the normal control group, the model control group showed significantly increased WBC, ALT, AST, and BUN (P≤0.05 or P≤0.01), and significantly decreased RBC (P≤0.05). Compared with the model control group, mice in the radiotherapy combined with low- and high-dose GLP-1 groups, the Kanglaite soft capsule group, and the Kanglaite soft capsule group showed significantly decreased blood WBC after administration (P≤0.05 or P≤0.01); mice in the radiotherapy combined with low-dose GLP-1 group showed significantly increased blood RBC (P≤0.05 or P≤0.01); mice in the Kanglaite soft capsule group showed significantly decreased blood ALT (P≤0.05 or P≤0.01); mice in the radiotherapy combined with low- and high-dose GLP-1 groups, the Kanglaite soft capsule group, and the Kanglaite soft capsule group showed significantly decreased BUN (P≤0.05); and mice in the radiotherapy combined with Kanglaite soft capsule group showed significantly decreased blood CRE (P≤0.05). Compared with the radiotherapy group, mice treated with radiotherapy combined with low- and high-dose GLP-1 and the Kanglaite soft capsule group showed significantly decreased blood WBC counts after administration (P≤0.05 or P≤0.01); mice treated with radiotherapy combined with low-dose GLP-1 showed significantly increased blood RBC counts after administration (P≤0.05 or P≤0.01); and mice treated with radiotherapy combined with Kanglaite soft capsule group showed significantly decreased blood CRE levels after administration (P≤0.05). Compared with the Kanglaite soft capsule group, mice treated with radiotherapy combined with low- and high-dose GLP-1 and the Kanglaite soft capsule group showed significantly decreased blood WBC counts after administration (P≤0.05 or P≤0.01); and mice treated with radiotherapy combined with low-dose GLP-1 and the Kanglaite soft capsule group showed significantly decreased blood AST levels after administration (P≤0.05 or P≤0.01). There were no statistically significant differences among the groups compared with the radiotherapy combined with Kanglaite soft capsule group.

[0333] Table 18 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on blood biochemical parameters in Lewis cell tumor-bearing mice (Mean±SEM)

[0334]

[0335] 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 Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01; compared with the radiotherapy + Kanglaite soft capsule group, ■ P≤0.05.

[0336] Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on CD3 in Lewis cell tumor-bearing mice + / CD4+ CD3 + / CD8 + Effect of content

[0337] As shown in Table 19, compared with the normal control group, the CD3 content of mice in the model control group was significantly higher. + / CD8 + Significantly increased (P≤0.05). Compared with the model control group, the radiotherapy combined with Kanglaite soft capsule group showed significantly higher CD3 levels in mice. + / CD4 + Significantly reduced (P≤0.05); In the radiotherapy combined with Kanglaite soft capsule group, the CD3 levels in mice were significantly reduced (P≤0.05). + / CD8 + Significantly reduced (P≤0.05). Compared with the radiotherapy group, the radiotherapy combined with Kanglaite soft capsule group showed significantly lower CD3 levels in mice. + / CD4 + Significantly reduced (P≤0.05); In the radiotherapy combined with Kanglaite soft capsule group, the CD3 levels in mice were significantly reduced (P≤0.05). + / CD8 + Significantly reduced (P≤0.05). No significant differences were found in other groups.

[0338] Table 19. Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on CD3 in Lewis cell tumor-bearing mice. + / CD4 + CD3 + / CD8 + Effect of content (Mean±SEM)

[0339]

[0340] Note: Compared with the normal control group, + P≤0.05; compared with the model control group, *P≤0.05; compared with the radiotherapy group, # P≤0.05, ## P≤0.01; compared with the Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01; compared with the radiotherapy + Kanglaite soft capsule group, ■ P≤0.05.

[0341] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on the histopathology of thymus, spleen, nose, stomach, and kidney tissues in Lewis cell tumor-bearing mice.

[0342] like Figure 26 , 27Results 28, 29, and 30 show that in the model control group, the spleen showed diffuse red pulp hematopoiesis and increased extramedullary hematopoiesis. In the radiotherapy group, the number of thymic lymphocytes decreased, and the medulla was diffuse; extramedullary hematopoiesis in the spleen's red pulp increased, while the number of white pulp lymphocytes decreased; multiple hemorrhages were observed in the nasal turbinate lamina propria. After radiotherapy combined with Ganoderma lucidum polysaccharide GLP-1, the number of thymic medullary lymphocytes decreased, while the number of splenic white pulp plasma cells increased; no abnormal lesions were observed in the nasal turbinates. No abnormal lesions were observed in the stomach and kidneys in any of the treatment groups.

[0343] like Figure 26 , 27 As shown in Figures 28, 29, and 30, the control groups are labeled as follows: A: Normal control group; B: Model control group; C: Radiotherapy group; E: Kanglaite soft capsule group; F: Radiotherapy + Kanglaite soft capsule group; G: Radiotherapy + GLP-1 low-dose group; H: Radiotherapy + GLP-1 high-dose group.

[0344] in conclusion

[0345] Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy can significantly inhibit the growth of tumors in Lewis lung cancer-bearing mice and has a significant synergistic effect.

[0346] Discussion and Summary

[0347] In recent years, with the gradual deterioration of the environment and factors such as smoking, the incidence of lung cancer has been rising year by year, with a high mortality rate and a trend towards affecting younger people. Clinically, the main treatment methods are surgery, radiotherapy, and chemotherapy to achieve early tumor eradication and reduce tumor burden. However, due to the highly invasive, metastatic, and insensitive nature of lung cancer cells to radiotherapy and chemotherapy, patients often experience immunosuppression after surgery, which accelerates tumor metastasis and development, leading to poor clinical prognosis.

[0348] The results of this experiment showed that the tumor volume of mice in the model control group was significantly increased, the thymus index was significantly decreased, the spleen index was significantly increased, the number of red blood cells in the blood was significantly increased, the number of white blood cells was significantly decreased, and liver and kidney function was significantly abnormal. In addition, CD4 + Increased content, CD8 +Significantly decreased, indicating a decline in the function of the lymphatic system in mice of the model control group during tumorigenesis. In the radiotherapy group, the tumor volume, body weight, etc. of the mice were significantly decreased, and the mortality rate was 40%. The results of this experiment showed that Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy could significantly inhibit the growth of tumors in mice, the thymus index and spleen index were significantly decreased, the number of red blood cells in the blood of mice was significantly decreased, the number of white blood cells was significantly increased, and the liver and kidney function indexes were decreased; the histopathological results showed that Ganoderma lucidum polysaccharide GLP-1 could increase the number of plasma cells in the white pulp of the spleen. The results showed that after GLP-1 was combined with radiotherapy, it could protect immune organs, enhance the body's autoimmune function, and thus play a role in reducing toxicity and enhancing anti-tumor effects. Compared with the Kanglaite soft capsule group and the radiotherapy combined with Kanglaite soft capsule group, Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy could significantly inhibit the growth of tumors, and the inhibitory effect of Ganoderma lucidum polysaccharide GLP-1 on tumor growth was significantly stronger than that of Kanglaite soft capsule.

[0349] In summary, Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy can significantly inhibit the growth of tumors in Lewis lung cancer-bearing mice and has a significant synergistic effect.

[0350] Experiment on the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on H22 liver cancer-bearing mice and data

[0351] Experimental purpose

[0352] Using C57 mice inoculated with homogenate of H22 liver cancer tumor mass under the right axilla to prepare tumor-bearing mice, to study the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-1 on liver cancer-bearing mice, and to provide experimental basis for its clinical research.

[0353] Experimental materials

[0354] Test samples

[0355] Ganoderma lucidum polysaccharide GLP-1, batch number: H2202208151A1; provided by Shenzhen Aolimei Tumor Medical Technology Co., Ltd.

[0356] Positive control

[0357] Cisplatin, batch number: E21268081, product of Shanghai Aladdin Biochemical Technology Co., Ltd.

[0358] Experimental animals

[0359] 80 SPF-grade male C57 mice, weighing 14 - 16 g, provided by Guangdong Medical Experimental Animal Center, experimental animal production license number: SCXK(Yue)2022 - 0002, experimental animal quality certificate number: 44007200107158.

[0360] Main reagents

[0361] 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.

[0362] Main instruments

[0363] Vernier calipers, Shanghai Tool Factory Co., Ltd.; I-2000 balance, Dongguan Nancheng Changxie Electronic Products Factory; Ophthalmic scissors and forceps, Shanghai Jinzhong Medical Instrument Co., Ltd.; CCL-170B-8 carbon dioxide incubator, Singapore ESCO Co., Ltd.; Luna-II cell counter, Nanjing Hengqiao Instrument Co., Ltd.

[0364] Experimental methods

[0365] A solid tumor model was established by injecting a homogenate suspension of H22 hepatocellular carcinoma into the subcutaneous tissue of the armpit of 65 healthy male C57 mice. When the average tumor volume of all mice reached approximately 230 mm³, they were randomly divided into groups based on tumor volume and administered the corresponding drugs or drug solvents via gavage or intraperitoneal injection, respectively, for 25 consecutive days. The long and short diameters of the tumors were measured every 3 days to calculate the tumor volume, and the mice were weighed every 3 days. At the end of the experiment, the tumors, spleen, and thymus of the mice were harvested and weighed, and the tumor index, spleen index, and thymus index were calculated.

[0366] Dosage design

[0367] Based on previous experimental results, Ganoderma lucidum polysaccharide GLP-1 was designed with a low dose of 50 mg / kg and a high dose of 150 mg / kg, and the corresponding drugs were administered as shown in Table 20.

[0368] The cisplatin dosage design was based on the clinical dosage of cisplatin not exceeding 100 mg / m2 per person per day and the tolerance of mice to cisplatin, and a dosage of 3 mg / kg was selected as the dosing dose.

[0369] Table 20 Trial grouping and dosage design

[0370]

[0371] detection indicators

[0372] therapeutic indicators

[0373] Relative tumor inhibition rate

[0374] Relative tumor inhibition rate (%) = (1-T) RTV / C RTV )×100%. Where, T RTV C represents the relative tumor volume of the experimental group. RTVThis represents the relative tumor volume of the model control group. Relative tumor volume RTV = V t / V0, V t Vt represents the tumor volume of mice on day t after drug administration, and V0 represents the tumor volume of mice at the time of grouping. Evaluation criteria: A relative tumor inhibition rate ≥40%, and statistical analysis showing P<0.05, is considered an effective inhibition level.

[0375] Tumor growth inhibition rate

[0376] Tumor growth inhibition rate (%) = (1-T / C)×100%. Where T represents the average tumor weight in the treatment group, and C represents the average tumor weight in the model control group. Evaluation criteria: A tumor growth inhibition rate ≥40% and statistical analysis P<0.05 were considered effective inhibition levels.

[0377] Spleen and thymus organ coefficients: After the last administration, the spleen, thymus, and tumor were weighed and the organ coefficients were calculated.

[0378] Tumor index (%) = tumor weight / body weight × 100%.

[0379] Immune organ index (mg / g) = organ mass / body weight × 1000.

[0380] Data processing and statistical analysis

[0381] SPSS 17.0 was used for statistical analysis. The level of statistical significance was set at P ≤ 0.05. Quantitative data were expressed as mean ± standard deviation (x ± s). Leven's test was used to test for normality and homogeneity of variance. If normality and homogeneity of variance were met (P > 0.05), one-way 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 (a non-parametric method) was used for comparative analysis. Statistical differences and biological significance were considered in the evaluation.

[0382] Experimental results

[0383] Animal mortality

[0384] As shown in Table 21, the mortality rate of mice in each group was 0.

[0385] Table 21 Statistics on the number of surviving animals and mortality rate in each group

[0386]

[0387] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on body weight of H22 hepatocellular carcinoma-bearing mice

[0388] As shown in Table 22, compared with the normal group, the body weight of mice in the model control group was significantly increased from D21 to D25, while the body weight of mice in the cisplatin group was significantly decreased from D18 to D25. The body weight of mice in the low-dose cisplatin + GLP-1 group was significantly decreased from D15 to D25, and the body weight of mice in the high-dose cisplatin + GLP-1 group was significantly decreased from D12 to D25. Compared with the model control group, the body weight of mice in the cisplatin group and the low-dose cisplatin + GLP-1 group was significantly decreased from D9 to D25; the body weight of mice in the high-dose cisplatin + GLP-1 group was significantly decreased from D6 to D25. Compared with the cisplatin group, the body weight of mice in the high-dose cisplatin + GLP-1 group was significantly decreased at D25.

[0389] Table 22 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on body weight in H22 liver cancer-bearing mice

[0390]

[0391] Note: Compared with the normal control group, + P≤0.05; compared with the model control group, * p<0.05; compared with the cisplatin group, # P<0.05.

[0392] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on tumor volume in H22 hepatocellular carcinoma-bearing mice

[0393] As shown in Table 23, compared with the model control group, the tumor volume was significantly reduced in the cisplatin group, the low-dose cisplatin + GLP-1 group, and the high-dose cisplatin + GLP-1 group from D3 to D25.

[0394] Compared with the cisplatin group, the tumor volume was significantly reduced in the low-dose cisplatin + GLP-1 group and the high-dose cisplatin + GLP-1 group from D15 to D25.

[0395] Table 23 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on tumor volume in H22 hepatocellular carcinoma-bearing mice

[0396]

[0397] Note: Compared with the model control group, * P<0.05; compared with the cisplatin group, # P<0.05.

[0398] Effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on relative tumor inhibition rate in H22 hepatocellular carcinoma-bearing mice

[0399] As shown in Table 24, compared with the model control group, the relative tumor inhibition rate of the cisplatin group was above 60% from day 12, reaching a maximum of 86.6%. The relative tumor inhibition rate of the low-dose and high-dose cisplatin + GLP-1 groups was above 40% from day 9, reaching a maximum of 94.3%. Compared with the cisplatin group, the relative tumor inhibition rate of the low-dose cisplatin + GLP-1 group was 30.4%–54.8% from day 15 to day 25 (P<0.05), and the relative tumor inhibition rate of the high-dose cisplatin + GLP-1 group was 52.4%–69.0% from day 15 to day 25 (P<0.05).

[0400] Table 24 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on relative tumor inhibition rate in H22 hepatocellular carcinoma-bearing mice (compared to the model control group)

[0401]

[0402] Note: Compared with the model control group, * P<0.05.

[0403] Table 25 Effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on relative tumor inhibition rate in H22 hepatocellular carcinoma-bearing mice (compared to cisplatin group)

[0404]

[0405] Note: Compared with the cisplatin group. # P<0.05.

[0406] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on organ coefficient and tumor growth inhibition rate in H22 hepatocellular carcinoma-bearing mice

[0407] As shown in Table 26, compared with the model group, the tumor index, spleen index, and thymus index were significantly reduced in the cisplatin group and the cisplatin combined with GLP-1 group. Compared with the cisplatin group, the tumor index was significantly reduced in the high-dose cisplatin + GLP-1 group, while there were no significant differences in the spleen index and thymus index in the cisplatin combined with GLP-1 group. Compared with the normal group, the thymus index was significantly reduced in the cisplatin combined with GLP-1 group.

[0408] Compared with the model control group, the tumor growth inhibition rate in the cisplatin group was 84.2%, while the rates in the low-dose and high-dose cisplatin + GLP-1 groups were 88.2% and 92.8%, respectively. Compared with the cisplatin group, the tumor growth inhibition rates in the low-dose and high-dose cisplatin + GLP-1 groups were 25.3% and 54.4%, respectively.

[0409] Table 26 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on organ index and tumor growth inhibition rate in H22 liver cancer-bearing mice

[0410]

[0411]

[0412] Note: Compared with the normal control group, + P≤0.05; compared with the model control group, * P<0.05; compared with the cisplatin group, # P<0.05.

[0413] like Figure 31 , 32 Images 33 and 34 show tumor-bearing mice, corresponding to the following groups: Figure 31 Model control group Figure 32 Cisplatin group, Figure 33 : Cisplatin + GLP-1 low-dose group Figure 34 Cisplatin + GLP-1 high-dose group

[0414] like Figure 35 , 36 Images 37 and 38 show tumor images in tumor-bearing mice, corresponding to the following groups: Figure 35 Model control group Figure 36 Cisplatin group, Figure 37 : Cisplatin + GLP-1 low-dose group Figure 38 Cisplatin + GLP-1 high-dose group

[0415] in conclusion

[0416] Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin can significantly inhibit the growth of tumors in H22 liver cancer-bearing mice and has a significant synergistic effect.

[0417] Experimental data on the antitumor effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on 4T1 breast cancer-bearing mice

[0418] Experimental Objective

[0419] Tumor-bearing mice were prepared by subcutaneously inoculating 4T1 breast cancer cells into the left shoulder and back of BALB / c mice. The anti-tumor effect of Ganoderma lucidum polysaccharide on breast cancer-bearing mice was studied to provide experimental evidence for its clinical research.

[0420] Experimental materials

[0421] Test sample

[0422] Ganoderma lucidum polysaccharide GLP-1, batch number: H20220706A; provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0423] Positive control

[0424] Cisplatin, batch number: E21268081, product of Shanghai Aladdin Biochemical Technology Co., Ltd.

[0425] Laboratory animals

[0426] 70 SPF-grade female BALB / c mice, weighing 14-16 g, were provided by Guangdong Medical Laboratory Animal Center. The production license number of laboratory animals: SCXK(Yue)2022-0002, and the quality certificate number of laboratory animals: 44007200113250.

[0427] Main reagents

[0428] PBS buffer solution was prepared by Shenzhen Aolimei Tumor Medical Technology Co., Ltd.; fetal bovine serum, a product of Zhejiang Tianhang Biotechnology Co., Ltd.; 1640 medium, a product of Gibco; 0.25% trypsin, a product of Gibco.

[0429] Main instruments

[0430] Vernier caliper, a product of Shanghai Tool Works Co., Ltd.; I-2000 type balance, Dongguan Nancheng Changxie Electronic Products Factory; ophthalmic scissors and ophthalmic forceps, products of Shanghai Jinzhong Medical Instruments Co., Ltd.; CCL-170B-8 carbon dioxide incubator, a product of ESCO in Singapore; Luna-II cell counter, a product of Nanjing Hengqiao Instruments Co., Ltd.

[0431] Experimental methods

[0432] A suspension of 4T1 breast cancer cells was injected subcutaneously into the left shoulder and back of 60 healthy female BALB / c mice to establish a solid tumor model. When the average tumor volume of all mice was about 190 mm3, they were randomly grouped according to the tumor volume and given the corresponding drugs or drug solvents by gavage or intraperitoneal injection for 18 consecutive days. The long and short diameters of the tumors were measured once every 3 days to calculate the tumor volume, and the body weights of the mice were weighed once every 3 days. At the end of the experiment, the tumors, spleens and thymuses of the mice were weighed, and the tumor index, spleen index and thymus index were calculated.

[0433] Dose design

[0434] Based on previous experimental results, Ganoderma lucidum polysaccharide GLP-1 was designed with a low dose of 50 mg / kg and a high dose of 150 mg / kg, and the corresponding drugs were given as shown in Table 27.

[0435] The basis for the cisplatin dosing design: 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 dosing dose.

[0436] Table 27 Experimental grouping and dose design

[0437]

[0438]

[0439] detection indicators

[0440] therapeutic indicators

[0441] Relative tumor inhibition rate

[0442] Relative tumor inhibition rate (%) = (1-T) RTV / C RTV )×100%. Where, T RTV C represents the relative tumor volume of the experimental group. RTV This represents the relative tumor volume of the model control group. Relative tumor volume RTV = V t / V0, V t Vt represents the tumor volume of mice on day t after drug administration, and V0 represents the tumor volume of mice at the time of grouping. Evaluation criteria: A relative tumor inhibition rate ≥40%, and statistical analysis showing P<0.05, is considered an effective inhibition level.

[0443] Tumor growth inhibition rate

[0444] Tumor growth inhibition rate (%) = (1-T / C)×100%. Where T represents the average tumor weight in the treatment group, and C represents the average tumor weight in the model control group. Evaluation criteria: A tumor growth inhibition rate ≥40% and statistical analysis P<0.05 were considered effective inhibition levels.

[0445] Spleen and thymus organ coefficients: After the last administration, the spleen, thymus, and tumor were weighed and the organ coefficients were calculated.

[0446] Tumor index (%) = tumor weight / body weight × 100%.

[0447] Immune organ index (mg / g) = organ mass / body weight × 1000.

[0448] Data processing and statistical analysis

[0449] SPSS 17.0 was used for statistical analysis. The level of statistical significance was set at P ≤ 0.05. Quantitative data were expressed as mean ± standard deviation (x ± s). Leven's test was used to test for normality and homogeneity of variance. If normality and homogeneity of variance were met (P > 0.05), one-way 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 (a non-parametric method) was used for comparative analysis. Statistical differences and biological significance were considered in the evaluation.

[0450] Experimental results

[0451] Animal mortality

[0452] As shown in Table 28, the mortality rate of mice in the model control group, cisplatin group, and cisplatin + GLP-1 low-dose group was 12.5%, while the mortality rate of mice in the other groups was 0.

[0453] Table 28 Statistics on the number of surviving animals and mortality rate in each group

[0454]

[0455] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on body weight in 4T1 breast cancer-bearing mice

[0456] As shown in Table 29, compared with the normal group, the body weight of mice in the model control group was significantly increased from D6 to D18, the body weight of mice in the low-dose cisplatin + GLP-1 group was significantly decreased from D6 to D18, and the body weight of mice in the cisplatin group and the high-dose cisplatin + GLP-1 group was significantly decreased from D3 to D18. Compared with the model control group, the body weight of mice in the cisplatin group, the low-dose cisplatin + GLP-1 group, and the high-dose cisplatin + GLP-1 group was significantly decreased from D3 to D18. Compared with the cisplatin group, the body weight of mice in the high-dose cisplatin + GLP-1 group was significantly decreased on D6.

[0457] Table 29 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on body weight in 4T1 breast cancer-bearing mice

[0458]

[0459] Note: Compared with the normal control group, + P≤0.05; compared with the model control group, * P<0.05; compared with the cisplatin group, # P<0.05.

[0460] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on tumor volume in 4T1 breast cancer-bearing mice

[0461] As shown in Table 30, compared with the model control group, the tumor volume was significantly reduced in the cisplatin group, the low-dose cisplatin + GLP-1 group, and the high-dose cisplatin + GLP-1 group from D3 to D18.

[0462] Compared with the cisplatin group, the low-dose cisplatin + GLP-1 group showed a significant reduction in tumor volume from day 3 to day 12, while the high-dose cisplatin + GLP-1 group showed a significant reduction in tumor volume from day 6 to day 18.

[0463] Table 30 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on tumor volume in 4T1 breast cancer-bearing mice

[0464]

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

[0466] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on relative tumor inhibition rate in 4T1 breast cancer-bearing mice

[0467] As shown in Table 31, compared with the model control group, the relative tumor inhibition rate of the cisplatin group reached more than 40% from day 9, and reached a maximum of 62.8% on day 18. The relative tumor inhibition rate of the cisplatin + GLP-1 low-dose group was more than 40% from day 3 to day 18, and the relative tumor inhibition rate of the cisplatin + GLP-1 high-dose group was more than 55% from day 6 to day 18, with the highest being 80.0% on day 18.

[0468] Compared with the cisplatin group, the relative tumor inhibition rate of the low-dose cisplatin + GLP-1 group was 23%–35% on days 3–12 (P<0.05), and the relative tumor inhibition rate of the high-dose cisplatin + GLP-1 group was 37%–53% on days 6–18 (P<0.05).

[0469] Table 31 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on relative tumor inhibition rate in 4T1 breast cancer-bearing mice (compared to the model control group)

[0470]

[0471] Note: Compared with the model control group, * P<0.05.

[0472] Table 32 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on relative tumor inhibition rate in 4T1 breast cancer-bearing mice (compared to the cisplatin group)

[0473]

[0474] Note: Compared with the cisplatin group. # P<0.05.

[0475] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on organ coefficient and tumor growth inhibition rate in 4T1 breast cancer-bearing mice

[0476] As shown in Table 33, compared with the model group, the tumor index, spleen index, and thymus index were significantly reduced in the cisplatin group and the cisplatin combined with GLP-1 group. Compared with the cisplatin group, the tumor index was significantly reduced in the high-dose cisplatin + GLP-1 group, while there were no significant differences in the spleen index and thymus index in the cisplatin combined with GLP-1 group. Compared with the normal group, the thymus index was significantly reduced in the model control group and the cisplatin combined with GLP-1 group, while the spleen index was significantly increased in the model control group.

[0477] Compared with the model control group, the tumor growth inhibition rate in the cisplatin group was 71.6%, while the rates in the low-dose and high-dose cisplatin + GLP-1 groups were 74.1% and 82.9%, respectively. Compared with the cisplatin group, the tumor growth inhibition rates in the low-dose and high-dose cisplatin + GLP-1 groups were 8.6% and 39.7%, respectively.

[0478] Table 33 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on organ index and tumor growth inhibition rate in 4T1 breast cancer-bearing mice

[0479]

[0480] Note: Compared with the normal control group, + P≤0.05; compared with the model control group, *P<0.05; compared with the cisplatin group, # P<0.05.

[0481] like Figure 39 , 40 Images 41 and 42 show tumor-bearing mice, corresponding to the following groups: Figure 39 Model control group Figure 40 Cisplatin group, Figure 41 : Cisplatin + GLP-1 low-dose group Figure 42 Cisplatin + GLP-1 high-dose group

[0482] like Figure 43 , 44 Images 45 and 46 show tumor images of tumor-bearing mice, corresponding to the following groups: Figure 43 Model control group Figure 44 Cisplatin group, Figure 45 : Cisplatin + GLP-1 low-dose group Figure 46 Cisplatin + GLP-1 high-dose group

[0483] in conclusion

[0484] Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin significantly inhibited tumor growth in 4T1 breast cancer-bearing mice and exhibited a significant synergistic effect.

[0485] Experimental data on the killing effect of Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin on LLC lung cancer cells

[0486] Experimental objective:

[0487] To investigate the killing effect of Ganoderma lucidum polysaccharide GLP-1 on LLC lung cancer cells and provide experimental evidence for its clinical research.

[0488] Experimental materials

[0489] Test sample

[0490] Ganoderma lucidum polysaccharide GLP-1; batch number: H20220319A, provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0491] Positive control

[0492] Cisplatin, batch number: E2128081, product of Shanghai Aladdin Biochemical Technology Co., Ltd.

[0493] cell lines

[0494] LLC lung cancer cell line was purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0495] Main reagents

[0496] PBS buffer was prepared by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.; 0.25% trypsin was a product of Gibco; MTT was a product of Aladdin; DMSO was a product of Guangdong Guanghua Technology Co., Ltd.; and DMEM medium was a product of Gibco.

[0497] Main instruments

[0498] 20μL, 200μL, and 1mL pipettes, products of Mettler Toledo Ltd.; Luna-II cell counter, product of Nanjing Hengqiao Instrument Co., Ltd.; ST-360 microplate reader, product of Shanghai Kehua Experimental System Co., Ltd.

[0499] Experimental methods:

[0500] LLC lung cancer cells were seeded into 96-well cell culture plates, with 3000 cells per well. After culturing at 37°C in a 5% CO2 incubator for 24 hours, 2 mg / ml GLP-1 solution was added to the test sample wells, and 2.5 μg / ml cisplatin solution was added to the positive wells. After culturing for another 48 hours, the absorbance at 570 nm was measured.

[0501] Test results

[0502] Table 34

[0503]

[0504] like Figure 47The figure shows the distribution of LLC lung cancer cells after 24 hours of culture.

[0505] like Figure 48 The figure shows the distribution of LLC lung cancer cells after 48 hours of culture.

[0506] like Figure 49 The figure shows the distribution of LLC lung cancer cells 24 hours after cisplatin administration.

[0507] like Figure 50 The figure shows the distribution of LLC lung cancer cells 48 hours after cisplatin administration.

[0508] like Figure 51 The figure shows the distribution of LLC lung cancer cells 24 hours after cisplatin + GLP-1 administration.

[0509] like Figure 52 The figure shows the distribution of LLC lung cancer cells 48 hours after administration of cisplatin + GLP-1.

[0510] in conclusion

[0511] Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin can inhibit the growth of LLC lung cancer cells and enhance the anti-tumor efficacy of cisplatin.

[0512] 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 within the protection scope of the present invention.

Claims

1. Ganoderma lucidum polysaccharide GLP-1, characterized in that, Molecular structural formula of the ganoderma lucidum polysaccharide GLP-1 ; molecular formula (C 60 H 100 O 50 )n; the main chain of the ganoderma lucidum polysaccharide GLP-1 is beta-1,4 glucan, and the beta-D-Glcp-(1→3,4)-beta-D-Glcp-(1→o-3 and→4,6)-beta-DGlcp-(1→o-6 bonds are connected on the main chain, and the molecular simplified formula of the ganoderma lucidum polysaccharide GLP-1 is ; wherein n=17-25.

2. Ganoderma lucidum polysaccharide GLP-1 according to claim 1, characterized in that, n is 17, 18, 19, 20, 21, 22, 23, 24, or 25.

3. The method for extracting Ganoderma lucidum polysaccharide GLP-1 according to claim 1 or 2, characterized in that, The method comprises the following steps: S1, remove the surface dust of Ganoderma lucidum by flushing with clean water, and crush the Ganoderma lucidum after drying at 105 DEG C into Ganoderma lucidum powder with a particle size greater than 60 mesh; S2, mix the crushed Ganoderma lucidum powder with water in a sealed container and heat, and fully mix the Ganoderma lucidum powder with water under high temperature and high pressure to form a medicinal juice solution; S3, use membrane concentration technology to separate the medicinal juice solution to obtain a concentrated solution with medicinal components and incomplete fusion residues; S4, mix the concentrated solution with medicinal components with pure water to obtain a water solution with a predetermined concentration by using multiple column chromatography separation technology, and obtain Ganoderma lucidum polysaccharide GLP-1 with medicinal components.

4. The method of claim 3, wherein the extraction of Ganoderma lucidum polysaccharide GLP-1 is characterized by, In step S2, the Ganoderma lucidum powder in the sealed container is mixed with water and fully stirred, and then heated to 105-200 DEG C under high temperature, and the boiling time is 2-6h, and the internal pressure of the sealed container gradually increases to form a high temperature and high pressure environment in the sealed container as the heating temperature increases.

5. The method of claim 4, wherein the extraction of Ganoderma lucidum polysaccharide GLP-1 is characterized by, In step S2, the mixed liquid of Ganoderma lucidum powder and water in the sealed container is heated to 105-170 DEG C under high temperature, and the boiling time is 3-6h, and the internal pressure of the sealed container gradually increases to form a high temperature and high pressure environment in the sealed container as the heating temperature increases. 6.The method of claim 5, wherein the extraction of the Ganoderma lucidum polysaccharide GLP-1 is characterized by, In step S4, the concentrated solution with medicinal components is mixed with pure water to obtain a water solution with a concentration ratio of 1:2-1:

5.

7. The method of claim 6, wherein the extraction of Ganoderma lucidum polysaccharide GLP-1 is characterized by, In step S3, the water solution with medicinal components is removed by using membrane concentration technology to obtain a concentrated solution or paste with medicinal components. 8.The method of claim 7, wherein the extraction of the Ganoderma lucidum polysaccharide GLP-1 is characterized by, In step S2, the mixed liquid in the sealed container is heated to 105 DEG C, 110 DEG C, 115 DEG C, 120 DEG C, 125 DEG C, 130 DEG C, 135 DEG C, 145 DEG C, 155 DEG C, 160 DEG C, 165 DEG C or 170 DEG C under high temperature, and the boiling time is 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h, and the internal pressure of the sealed container gradually increases to form a high temperature and high pressure environment in the sealed container as the heating temperature increases. 9.The method of claim 4, wherein the extraction of the Ganoderma lucidum polysaccharide GLP-1 is characterized by, In step S2, the mixed liquid in the sealed container is heated to 105 DEG C, 110 DEG C, 115 DEG C, 120 DEG C, 125 DEG C, 130 DEG C, 135 DEG C, 145 DEG C, 155 DEG C, 160 DEG C, 165 DEG C, 170 DEG C, 175 DEG C, 180 DEG C, 185 DEG C, 190 DEG C, 195 DEG C or 200 DEG C under high temperature, and the boiling time is 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h, and the internal pressure of the sealed container gradually increases to form a high temperature and high pressure environment in the sealed container as the heating temperature increases.

10. The use of Ganoderma lucidum polysaccharide GLP-1 according to claim 1 or 2, characterized in that, When the Ganoderma lucidum polysaccharide GLP-1 is combined with the chemotherapy drug cisplatin, the toxic side effects of the chemotherapy drug on the human body can be eliminated, and the control, reduction and elimination of tumor cells can be achieved.

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