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

Abstract

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

Description

A polysaccharide compound with clear molecular structure suitable for treating pulmonary fibrosis TECHNICAL FIELD

[0001] The present application belongs to the field of improvement of plant extraction and separation technology, and particularly relates to a method for extracting Ganoderma lucidum polysaccharide GLP-4 and application thereof. BACKGROUND

[0002] Ganoderma lucidum is a kind of fungal plant, and has a long history of medicinal use in China and Japan. There are many active ingredients in Ganoderma lucidum, and the composition is complex. In the past, more than 150 compounds have been isolated from Ganoderma lucidum, mainly including polysaccharides, triterpenes, sterols, alkaloids, furan derivatives, amino polypeptides and inorganic elements. Different geographical locations (longitude and latitude), different seeds, different growth environments, and different temperature, humidity and light intensity can have a significant impact on the content, proportion and presence or absence of the medicinal components mentioned in the present application in Ganoderma lucidum.

[0003] SUMMARY

[0004] The present application aims to provide a polysaccharide compound with clear molecular structure, which has the effects of treating emphysema, pulmonary fibrosis and anti-tumor.

[0005] The present application aims to solve the treatment problem of emphysema and pulmonary fibrosis which has not been solved internationally. At present, there is no specific drug for the treatment of emphysema and pulmonary fibrosis internationally. The emergence of Ganoderma lucidum polysaccharide compound GLP-4 is expected to fill this gap, and the compound also shows excellent effect on the treatment of late-stage cancer, especially when used in combination with chemotherapy drugs, which can eliminate the toxic and side effects of chemotherapy drugs on the human body. It also plays a role in preventing, controlling and treating pulmonary fibrosis and the transformation of pulmonary nodules into lung cancer.

[0006] The medicinal components mentioned in the present application also have an important function of preventing the variation of normal human cells and preventing the generation of cancer cells.

[0007] The present application is achieved by a method for extracting Ganoderma lucidum polysaccharide GLP-4, which comprises the following steps:

[0008] S1, drying and crushing Ganoderma lucidum to obtain Ganoderma lucidum powder;

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

[0010] S3, separating the medicinal juice solution by centrifugation to obtain a medicinal residue and a medicinal component concentrated solution;

[0011] S4, mixing the separated medicinal residue with NaOH solution according to a predetermined ratio, soaking, and then adding HCl for neutralization.

[0012] S5, concentrating and desalting the mixed solution by a membrane concentration technology to obtain a concentrated solution containing the medicinal components;

[0013] S6, preparing a concentrated solution containing medicinal components by freeze-drying the concentrated solution, and then preparing a solution with a certain concentration through multiple column chromatography to obtain ganoderma lucidum polysaccharide GLP-4 with medicinal components.

[0014] A further technical solution of the present application is that in the step S2, the ganoderma lucidum powder in the sealed container is mixed with water and stirred sufficiently, and then heated to 105-200 DEG C, and the boiling time lasts for 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.

[0015] A further technical solution of the present application is that in the step S2, the mixed liquid of the ganoderma lucidum powder and water in the sealed container is heated to 105-170 DEG C, and the boiling time lasts for 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.

[0016] A further technical solution of the present application is that in the step S6, the freeze-dried powder containing medicinal components is prepared into a solution with a concentration of 40-80mg / ml by adding pure water, and then separated through multiple column chromatography.

[0017] A further technical solution of the present application is that in the step S4, the medicinal residues are mixed with the NaOH solution at a temperature of 40-100 DEG C according to a ratio of 1:10-1:40, and the soaking time is 1-5h, wherein the concentration of NaOH is 0.05-0.5mol / L.

[0018] A further technical solution of the present application is that in the step S3, the extraction liquid is separated from the ganoderma lucidum residues by using a centrifugal technology to obtain the ganoderma lucidum residues, and the ganoderma lucidum residues are further extracted and purified.

[0019] A further technical solution of the present application is that in the step S1, the ganoderma lucidum is washed by clean water to remove the surface dust, and then dried at 105 DEG C, and the dried ganoderma lucidum is crushed, and the crushed ganoderma lucidum powder is greater than 60 mesh.

[0020] The further technical scheme of the present application is that in the step S2, the mixed liquid in the closed 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, 140 DEG C, 145 DEG C, 150 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, 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 closed container gradually increases with the increase of the heating temperature to form a high-temperature and high-pressure environment in the closed container.

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

[0022] The further technical scheme of the present application is that n is 10, 11, 12, 13, 14 or 15.

[0023] Another object of the present application is to provide an application of the ganoderma lucidum polysaccharide GLP-4, and the ganoderma lucidum polysaccharide GLP-4 has good water-soluble performance, is easy to be absorbed by human bodies, has the effects of treating emphysema, pulmonary fibrosis and resisting tumors, especially has an excellent treatment effect on emphysema and pulmonary fibrosis, is obviously superior to pirfenidone, and when the ganoderma lucidum polysaccharide GLP-4 is used in combination with a chemotherapy drug, the ganoderma lucidum polysaccharide GLP-4 can eliminate the toxic and side effects of the chemotherapy drug on human bodies, has a certain effect on controlling, reducing and eliminating tumor cells.

[0024] The ganoderma lucidum polysaccharide GLP-4 has the advantages that the extraction process is simple, the ganoderma lucidum residues are fully utilized, the production cost is low, the polysaccharide extraction rate is high, and the operation is simple. BRIEF DESCRIPTION OF DRAWINGS

[0025] Fig. 1 is a flow chart of a method for extracting the ganoderma lucidum polysaccharide GLP-4 according to an embodiment of the present application.

[0026] Fig. 2 is a correction curve diagram of lgMp-RT (peak molecular weight) according to an embodiment of the present application.

[0027] Fig. 3 is a correction curve diagram of lgMp-RT (weight average molecular weight) according to an embodiment of the present application.

[0028] Fig. 4 is a correction curve diagram of lgMp-RT (number average molecular weight) according to an embodiment of the present application.

[0029] Figure 5 is a schematic diagram of the molecular weight profile of Ganoderma lucidum polysaccharide GLP-4 according to an embodiment of the present application.

[0030] Figure 6 is a schematic diagram of the ion chromatogram of Mix 16 according to an embodiment of the present application.

[0031] Figure 7 is a schematic diagram of the ion chromatogram of Mix 16 according to an embodiment of the present application.

[0032] Figure 8 is a GCMS chromatogram of Ganoderma lucidum polysaccharide GLP-4 (PMAA) according to an embodiment of the present application.

[0033] Figure 9 is a schematic diagram of the result analysis of polysaccharide methylated glycol acetyl ester (PMAA) according to an embodiment of the present application.

[0034] Figure 10 is a schematic diagram of the result analysis of polysaccharide methylated glycol acetyl ester (PMAA) according to an embodiment of the present application.

[0035] Figure 11 is a schematic diagram of the result analysis of polysaccharide methylated glycol acetyl ester (PMAA) according to an embodiment of the present application.

[0036] Figure 12 is a schematic diagram of the result analysis of polysaccharide methylated glycol acetyl ester (PMAA) according to an embodiment of the present application.

[0037] Figure 13 is a schematic diagram of the result analysis of polysaccharide methylated glycol acetyl ester (PMAA) according to an embodiment of the present application.

[0038] Figure 14 is a schematic diagram of the result analysis of polysaccharide methylated glycol acetyl ester (PMAA) according to an embodiment of the present application.

[0039] Figure 15 is a schematic diagram of the hydrogen spectrum according to an embodiment of the present application.

[0040] Figure 16 is a schematic diagram of the carbon spectrum according to an embodiment of the present application.

[0041] Figure 17 is a schematic diagram of Dept 135 spectrum according to an embodiment of the present application.

[0042] Figure 18 is a schematic diagram of HH-COSY according to an embodiment of the present application.

[0043] Figure 19 is a schematic diagram of HSQC according to an embodiment of the present application.

[0044] Figure 20 is a schematic diagram of HMBC according to an embodiment of the present application.

[0045] Figure 21 is a schematic diagram of NOESY according to an embodiment of the present application.

[0046] Figure 22 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the tumor volume of Lewis cell tumor-bearing mice according to an embodiment of the present application.

[0047] Figure 23 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the pathology of tumor in Lewis cell tumor-bearing mice (x200) according to an embodiment of the present application.

[0048] Figure 24 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the pathology of kidney in Lewis cell tumor-bearing mice (x200) according to an embodiment of the present application.

[0049] Figure 25 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the pathology of stomach in Lewis cell tumor-bearing mice (x200) according to an embodiment of the present application.

[0050] Figure 26 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the pathology of spleen in Lewis cell tumor-bearing mice (x200) according to an embodiment of the present application.

[0051] Figure 27 is a schematic diagram of the control group of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 hepatocarcinoma tumor-bearing mouse model according to an embodiment of the present application.

[0052] Figure 28 is a schematic diagram of the cisplatin group of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 hepatocarcinoma tumor-bearing mouse according to an embodiment of the present application.

[0053] Figure 29 is a schematic diagram of the cisplatin+GLP-4 low-dose group of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 hepatocarcinoma tumor-bearing mouse according to an embodiment of the present application.

[0054] Figure 30 is a schematic diagram of the cisplatin+GLP-4 high-dose group of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 hepatocarcinoma tumor-bearing mouse according to an embodiment of the present application.

[0055] Figure 31 is a schematic diagram of the control group of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 hepatocarcinoma tumor-bearing mouse tumor model according to an embodiment of the present application.

[0056] Figure 32 is a schematic diagram of the cisplatin group of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 hepatocarcinoma tumor-bearing mouse tumor according to an embodiment of the present application.

[0057] Figure 33 is a schematic diagram of the cisplatin+GLP-4 low-dose group of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 hepatocarcinoma tumor-bearing mouse tumor according to an embodiment of the present application.

[0058] Figure 34 is a schematic diagram of the cisplatin+GLP-4 high-dose group of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 hepatocarcinoma tumor-bearing mouse tumor according to an embodiment of the present application.

[0059] Figure 35 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 orthotopic hepatocarcinoma mouse tumor (binning: 8x8; T=30s) according to an embodiment of the present application.

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

[0061] Figure 37 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the liver of H22 orthotopic liver cancer in mice (x200) according to an embodiment of the present application.

[0062] Figure 38 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the spleen of H22 orthotopic liver cancer in mice (x200) according to an embodiment of the present application.

[0063] Figure 39 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the stomach of H22 orthotopic liver cancer in mice (x200) according to an embodiment of the present application.

[0064] Figure 40 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on the kidney of H22 orthotopic liver cancer in mice (x200) according to an embodiment of the present application.

[0065] Figure 41 is a confocal image of the RAW264.7 macrophage blank control group under 40x magnification according to an embodiment of the present application.

[0066] Figure 42 is a confocal image of 0.25 mg / mL Ganoderma lucidum polysaccharide GLP-4 incubated with RAW264.7 macrophages for 24 hours under 40x magnification according to an embodiment of the present application.

[0067] Figure 43 is a confocal image of 0.25 mg / mL Ganoderma lucidum polysaccharide GLP-4 incubated with RAW264.7 macrophages for 24 hours under 63x oil magnification according to an embodiment of the present application.

[0068] Figure 44 is a confocal image of 0.5 mg / mL Ganoderma lucidum polysaccharide GLP-4 incubated with RAW264.7 macrophages for 24 hours under 40x magnification according to an embodiment of the present application.

[0069] Figure 45 is a confocal image of 0.5 mg / mL Ganoderma lucidum polysaccharide GLP-4 incubated with RAW264.7 macrophages for 24 hours under 63x oil magnification according to an embodiment of the present application.

[0070] Figure 46 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-4 combined with cisplatin on the necrosis rate and apoptosis rate of H22 cells according to an embodiment of the present application.

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

[0072] Figure 48 is a schematic diagram of the cisplatin group of 4T1 breast cancer tumor-bearing mice treated with Ganoderma lucidum polysaccharide GLP-4 in combination with chemotherapy according to an embodiment of the present application.

[0073] Figure 49 is a schematic diagram of the cisplatin+GLP-4 low-dose group of 4T1 breast cancer tumor-bearing mice treated with Ganoderma lucidum polysaccharide GLP-4 in combination with chemotherapy according to an embodiment of the present application.

[0074] Figure 50 is a schematic diagram of the cisplatin+GLP-4 high-dose group of 4T1 breast cancer tumor-bearing mice treated with Ganoderma lucidum polysaccharide GLP-4 in combination with chemotherapy according to an embodiment of the present application.

[0075] Figure 51 is a schematic diagram of the tumor model control group of 4T1 breast cancer tumor-bearing mice treated with Ganoderma lucidum polysaccharide GLP-4 in combination with chemotherapy according to an embodiment of the present application.

[0076] Figure 52 is a schematic diagram of the tumor cisplatin group of 4T1 breast cancer tumor-bearing mice treated with Ganoderma lucidum polysaccharide GLP-4 in combination with chemotherapy according to an embodiment of the present application.

[0077] Figure 53 is a schematic diagram of the tumor cisplatin+GLP-4 low-dose group of 4T1 breast cancer tumor-bearing mice treated with Ganoderma lucidum polysaccharide GLP-4 in combination with chemotherapy according to an embodiment of the present application.

[0078] Figure 54 is a schematic diagram of the tumor cisplatin+GLP-4 high-dose group of 4T1 breast cancer tumor-bearing mice treated with Ganoderma lucidum polysaccharide GLP-4 in combination with chemotherapy according to an embodiment of the present application.

[0079] Figure 55 is a schematic diagram of the lung tissue pathology of normal group mice (x40) according to an embodiment of the present application.

[0080] Figure 56 is a schematic diagram of the lung tissue pathology of model control group mice (x40) according to an embodiment of the present application.

[0081] Figure 57 is a schematic diagram of the lung tissue pathology of positive control group mice (x40) according to an embodiment of the present application.

[0082] Figure 58 is a schematic diagram of the lung tissue pathology of Ganoderma lucidum polysaccharide GLP-4 low-dose group mice (x40) according to an embodiment of the present application.

[0083] Figure 59 is a schematic diagram of the lung tissue pathology of Ganoderma lucidum polysaccharide GLP-4 high-dose group mice (x40) according to an embodiment of the present application. DETAILED DESCRIPTION

[0084] Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein the same or like reference numerals are used throughout the drawings to refer to the same or like elements or elements with the same or similar functionality. The embodiments described below are illustrative examples of how to make and use the present application and are not intended to be limiting.

[0085] As shown in Figure 1, the present application provides a flow chart of the method for extracting Ganoderma polysaccharide GLP-4, which is described in detail as follows:

[0086] Step S1, the picked Ganoderma or the Ganoderma after the preliminary simple treatment is washed with clean water on a cleaning device to remove the floating dust on the surface of the Ganoderma, and the Ganoderma after the removal of the floating dust is transferred to a drying device for drying at a drying temperature of 105℃. The dried Ganoderma is crushed into Ganoderma powder on a crusher, and the Ganoderma powder is sieved. The Ganoderma powder particles larger than 60 mesh are converted into the next process, and the Ganoderma powder particles smaller than 60 mesh are returned to the crusher for re-crushing. The process is repeated for multiple times until the crushed Ganoderma powder particles meet the specified requirements.

[0087] Step S2, the Ganoderma powder meeting the requirements is mixed with pure water and placed in a sealed container. The sealed container is subjected to high-temperature heating. During the heating process, the internal pressure of the sealed container gradually increases with the increase of the temperature, so that a high-temperature and high-pressure environment is formed in the sealed container. In this environment, the Ganoderma powder and water are more easily fully mixed to form a mixed liquid. The sealed container is subjected to high-temperature heating to a boiling temperature of 105℃-200℃ for a boiling time of 2-6h. Preferably, the sealed container is subjected to high-temperature heating to a boiling temperature of 105℃-170℃ for a boiling time of 3-6h. More preferably, the sealed container is subjected to high-temperature heating to a boiling temperature of 105℃, 110℃, 115℃, 120℃, 125℃, 130℃, 135℃, 140℃, 145℃, 150℃, 155℃, 160℃, 165℃, 170℃, 175℃, 180℃, 185℃, 190℃, 195℃ or 200℃ for a boiling time of 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h, so that the mixed liquid is fully mixed to form an aqueous solution containing the medicinal components.

[0088] Step S3, the aqueous solution containing the medicinal components is separated from the Ganoderma dregs by using centrifugal technology. The Ganoderma extraction liquid and the Ganoderma dregs are obtained, and the Ganoderma extraction liquid is used for other medicinal research.

[0089] Step S4, the Ganoderma dregs obtained after the centrifugal separation are soaked with a NaOH solution according to a solid-liquid ratio of 1:10-1:40. The extraction temperature is 40-100℃, and the extraction time is 1-5h. The concentration of sodium hydroxide is 0.05-0.5mol / L. After the extraction is completed, hydrochloric acid is added for neutralization and centrifugal dreg removal to obtain a mixed solution.

[0090] Step S5, the mixed solution is concentrated and desalted by using membrane concentration technology to remove the NaCl components in the mixed solution, and a concentrated solution with medicinal components is obtained.

[0091] Step S6, the concentrated liquid containing the medicinal component is freeze-dried to obtain a powder, and then the powder is dissolved in pure water to obtain a solution with a concentration of 40-80 mg / ml; the solution is separated by multiple column chromatography, and then concentrated and freeze-dried to obtain the ganoderma lucidum polysaccharide GLP-4.

[0092] The method has simple extraction process, high polysaccharide extraction rate, is a reutilization of ganoderma lucidum residues, and has low production cost.

[0093] The present application aims to provide a ganoderma lucidum polysaccharide GLP-4, and the structural formula of the ganoderma lucidum polysaccharide GLP-4 is Molecular formula (C 162 H 270 O 135 ) n Wherein, n = 10-15.

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

[0095] After the ganoderma lucidum polysaccharide GLP-4 with the above structure is obtained, a determination experiment is performed, and the following report results are obtained.

[0096] II. Molecular weight determination

[0097] 1. Experimental purpose

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

[0099] 2. Experimental materials

[0100] 2.1 Instrument

[0101] 2.2 Materials

[0102] 2.3 Standard

[0103] 3. Experimental steps

[0104] 3.1 Reagent preparation

[0105] 3.2 Sample and standard solution preparation

[0106] The sample and the standard are accurately weighed, the sample is prepared into a 5mg / ml solution, centrifuged at 12000rpm for 10min, the supernatant is filtered with a 0.22mu m microporous filter, and then the sample is transferred to a 1.8ml sample vial.

[0107] 3.3 Chromatographic method

[0108] Chromatographic column: BRT 105-104-102 gel column (8 x 300 mm) in series; mobile phase: 0.05 M NaCl solution; flow rate: 0.6 ml / min, column temperature: 40 °C; sample injection volume: 20 μl; detector: differential refractometer RI-10A.

[0109] 4. Experimental results

[0110] As shown in Figures 2-4, lgMp-RT (peak molecular weight), lgMw-RT (weight average molecular weight), and lgMn-RT (number average molecular weight) calibration curves were obtained.

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

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

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

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

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

[0116] II. Determination of monosaccharide composition experiment

[0117] 1. Experimental purpose

[0118] The ion chromatograph was used to determine the monosaccharide composition.

[0119] 2. Experimental principle

[0120] Based on the fact that sugar molecules have electrochemical activity and are in an ionized state in strong alkali solution. Sugar compounds are weak acids with pKa > 11, and in high pH eluent, they exist in anionic form, partially or completely. According to the differences in ion exchange caused by the differences in pKa of different sugar compounds and the differences in hydrophobicity between certain sugar compounds and anion exchange resins, high-efficiency anion exchange separation of sugar compounds is realized, and then the current generated by the oxidation reaction of the hydroxyl group in the sugar molecule structure on the gold electrode surface is detected.

[0121] 3. Experimental materials

[0122] 3.1 Instruments

[0123] 3.2 Reagents

[0124] 3.3 Standards

[0125] 4. Experimental Methods

[0126] 4.1 Reagent Preparation

[0127] 4.2 Preparation of Standard Solution and Calculation Method

[0128] Take 16 kinds of monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid, mannuronic acid) to prepare standard stock solution.

[0129] Take each monosaccharide standard solution to precisely prepare concentration standards as a mixed standard. According to the absolute quantitative method, determine the mass of different monosaccharides, and calculate the molar ratio according to the molar mass of monosaccharides.

[0130] 4.3 Sample Preparation

[0131] Precisely weigh 5 mg of GLP-4 sample into an ampoule, add 3M TFA 2 ml, hydrolyze at 120°C for 3h. Accurately pipette the acid hydrolysis solution into a tube and dry it under nitrogen, add 5 ml of water and vortex to mix, pipette 50 uL into 950 uL of deionized water, centrifuge at 12000 rpm for 5 min. Take the supernatant for IC analysis.

[0132] 4.4 Chromatographic Method

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

[0134] 4.5 Standard Sequence

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

[0136] 5. Experimental Results

[0137] Mixed standard: solvent peak: 2.0 min for sodium hydroxide peak, 41 min for sodium acetate peak. As shown in Figures 6, 7.​

[0138] III. Determination of the linkage of Ganoderma lucidum polysaccharide GLP-4

[0139] 1. Purpose of the experiment

[0140] The linkage of Ganoderma lucidum polysaccharide GLP-4 was determined by GC-MS after methylation and other derivatization of the sample.

[0141] 2. Experimental materials

[0142] 2.1 Instruments

[0143] 2.2 Reagents

[0144] 3. Experimental methods

[0145] 3.1 Preparation of reagents

[0146] 3.2 Sample methylation

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

[0148] Ganoderma lucidum polysaccharide GLP-4 sample (2-3 mg) was weighed into a glass reaction bottle, 1 mL of anhydrous DMSO was added, and methylating reagent A solution was quickly added, sealed, dissolved under ultrasonic action, and then methylating reagent B solution was added. The reaction was carried out in a magnetic stirring water bath at 30°C for 60 min. Finally, 2 mL of ultrapure water was added to the above mixture to terminate the methylation reaction.

[0149] The methylated polysaccharide was taken and 1 ml of 2M trifluoroacetic acid (TFA) was added for hydrolysis for 90 min, and then rotary evaporation was performed to dryness. The residue was added with 2 ml of double-distilled water and 60 mg of sodium borohydride was added for reduction for 8 hours. Glacial acetic acid was added for neutralization, and then rotary evaporation was performed. The sample was dried in an oven at 101°C, and then 1 ml of acetic anhydride was added for acetylation at 100°C for 1 h, and then cooled. Then 3 mL of toluene was added, and the sample was concentrated and dried under reduced pressure, and the operation was repeated 4-5 times to remove excess acetic anhydride.

[0150] The acetylated product was dissolved in 3 mL of CH2Cl2 and transferred to a separatory funnel. A small amount of distilled water was added, and the mixture was shaken thoroughly. The upper aqueous solution was removed, and the operation was repeated 4 times. The CH2Cl2 layer was dried with an appropriate amount of anhydrous sodium sulfate, and then the volume was adjusted to 10 mL, and the sample was placed in a liquid phase vial. The acetylated product sample was determined by Shimadzu GCMS-QP 2010 gas chromatography-mass spectrometry.

[0151] GC-MS conditions: RXI-5SIL MS chromatographic column 30 m*0.25 mm*0.25 um; the temperature program is as follows: the initial temperature is 120℃, and the temperature is increased to 250℃ / min at a rate of 3℃ / min; the temperature is maintained for 5 min; the injection port temperature is 250℃, the detector temperature is 250℃, the carrier gas is helium, and the flow rate is 1 mL / min.

[0152] 4. Experimental results

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

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

[0155] IV. NMR analysis spectrum and analysis

[0156] 1. Experimental materials and instruments

[0157] Heavy water (D2O, 99.9%), deuterated acetone as an internal reference; freeze dryer, Bruker 600M nuclear magnetic resonance instrument (Nuclear Magnetic Resonance, NMR);

[0158] 2. Experimental steps

[0159] 50 mg of the polysaccharide GLP-4 sample was weighed, dissolved in 0.5 ml of heavy water, and freeze-dried. Then the freeze-dried powder was dissolved in 0.5 ml of heavy water and freeze-dried again, and the above process was repeated to fully exchange the active hydrogen. Then the sample was dissolved in 0.5 ml of heavy water and measured at room temperature 25℃ on a nuclear magnetic resonance instrument at 600 MHz. 1 H NMR spectrum, 13 C NMR spectrum, DEPT135 one-dimensional spectrum, and two-dimensional spectrum.

[0160] 3. Experimental results

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

[0162] Carbon spectrum analysis is in 13C NMR (201 MHz, D20): The main signals of carbon spectrum were concentrated between 60-120 ppm. By observing the carbon spectrum, it can be seen that the main anomeric carbon signal peaks δ 104.02, 103.92, 103.91, 103.83, 103.66 anomeric carbon region is mainly between δ 93-105. While δ 71.47, 74.99, 69.23, 76.08, 62.09, 76.69, 74.09, 82.09, 76.43, 70.09, 74.61, 76.65, 76.01, 70.89, 70.13, 74.42, 85.43, 69.96, 77.31, 70.13, 74.16, 71.57, 82.28, 76.68, 61.66, 74.76, 85.64, 69.36, 77.11, 62.03 ppm region. According to the results of monosaccharide composition, the polysaccharide is composed of glucose. It is shown that the polysaccharide GLP-4 is mainly glucan. As shown in Figure 16.

[0163] Dept 135 map analysis, 60.70, 68.82, 61.66, 62.04, 70.28, 68.79 ppm peaks are inverted peaks, indicating the chemical shift of C6. As shown in Figure 17.

[0164] As shown in Figures 18-21, by HSQC spectrum, it can be observed that the anomeric carbon signal is δ 103.94, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ 4.68. By HH-COSY, the signal of H1-2 is 4.68 / 3.44; the signal of H2-3 is 3.44 / 3.65; the signal of H3-4 is 3.65 / 3.41; we can infer that H1, H2, H3, H4 are δ 4.68, 3.44, 3.65, 3.41 respectively. The corresponding δ 103.66, 74.76, 85.64, 69.36. The chemical shift of C5 is δ 77.11; the chemical shift of C6 is δ 62.03. Therefore, the signal should be attributed to the glycosidic bond→3)-β-Glcp-(1→.

[0165] By HSQC spectrum, the anomeric carbon signal can be observed as δ 103.91, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ 4.44. Through HH-COSY, the signal of H1-2 is 4.44 / 3.23; the signal of H2-3 is 3.23 / 3.41; the signal of H3-4 is 3.41 / 3.55; we can infer that H1, H2, H3, H4 are δ 4.44, 3.23, 3.41, 3.55, respectively, and the corresponding C1-4 are δ 103.92, 74.61, 76.65, 76.01; and the NOESY spectrum observes that δ 4.44 has correlation peaks with 3.23, 3.41, 3.55, 4.12, and Dept135 combined with HSQC can determine that δ 3.77, 4.12 are the peaks of H6a,b, and H5 is 3.36 ppm. The corresponding C5 is 70.89; the chemical shift of C6 is δ 70.13, and the corresponding H6a is δ 3.77, 4.12. Therefore, the signal should be attributed to the glycosidic bond →6)-β-Glcp-(1→.

[0166] According to similar rules and combined with HMBC and NOESY, the signals of all glycosidic bonds are attributed as follows:

[0167] Hydrogen and carbon signal attribution

[0168] Main chain analysis:

[0169] In the HMBC spectrum, according to the one-dimensional and two-dimensional nuclear magnetic resonance spectrum, the glycosidic bond signals of the polysaccharide GLP-4 are attributed; by analyzing the chemical shift of the hydrogen spectrum less than 5ppm, the monosaccharide composition is glucose, and therefore, it is β-glucan. The anomeric hydrogen of the glycosidic bond →6)-β-D-Glcp-(1→ has a correlation signal peak with its own C6; indicating the presence of the linkage mode of →6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→.

[0170] The anomeric hydrogen of the glycosidic bond →6)-β-D-Glcp-(1→ has a correlation signal peak with the C6 of its →3,6)-β-D-Glcp-(1→; indicating the presence of the linkage mode of →6)-β-D-Glcp-(1→3,6)-β-D-Glcp-(1→.

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

[0172] The anomeric hydrogen of the glycosidic bond → 3,6)-β-D-Glcp-(1→ has a correlation signal peak with C6 of → 4,6)-β-D-Glcp-(1→, indicating that there is a linkage mode of → 3,6)-β-D-Glcp-(1→ 4,6)-β-D-Glcp-(1→.

[0173] Branch analysis:

[0174] In the NOESY map, the anomeric hydrogen of the glycosidic bond β-D-Glcp-(1→ has a correlation signal peak with H3 of → 3)-β-D-Glcp-(1→, indicating that there is a linkage mode of β-D-Glcp-(1→ 3)-β-D-Glcp-(1→.

[0175] The anomeric hydrogen of the glycosidic bond → 3)-β-D-Glcp-(1→ has a correlation signal peak with H3 of → 3)-β-D-Glcp-(1→, indicating that there is a linkage mode of → 3)-β-D-Glcp-(1→ 3,6)-β-D-Glcp-(1→.

[0176] The anomeric hydrogen of the glycosidic bond → 4)-β-D-Glcp-(1→ has a correlation signal peak with H4 of → 4,6)-β-D-Glcp-(1→, indicating that there is a linkage mode of → 4)-β-D-Glcp-(1→ 4,6)-β-D-Glcp-(1→.

[0177] The anomeric hydrogen of the glycosidic bond → 4)-β-D-Glcp-(1→ has a correlation signal peak with H4 of → 4,6)-β-D-Glcp-(1→, indicating that there is a linkage mode of → 4)-β-D-Glcp-(1→ 4,6)-β-D-Glcp-(1→.

[0178] Based on the above, it can be inferred that the main chain of the polysaccharide GLP-4 is β-1,6 glucan, and β-D-Glcp-(1→ 3)-β-D-Glcp-(1→ and β-D-Glcp-(1→ 4)-β-D-Glcp-(1→ are connected to the main chain through O-3 of → 3,6)-β-D-Glcp-(1→ and O-4 of → 4,6)-β-D-Glcp-(1→ respectively, and the molecular simplification formula is as follows.

[0179] Another object of the present application is to provide a use of the ganoderma lucidum polysaccharide GLP-4, which has good water solubility and is easily absorbed by the human body, has the effects of treating emphysema, pulmonary fibrosis and anti-tumor, especially the treatment effects on emphysema and pulmonary fibrosis are exceptionally excellent, and are obviously superior to pirfenidone, and when combined with a chemotherapy drug, can eliminate the toxic and side effects of the chemotherapy drug on the human body and has a certain effect on the control, reduction and elimination of tumor cells.

[0180] The anti-tumor effect of Ganoderma lucidum polysaccharide GLP-4 is as follows:

[0181] Ganoderma lucidum polysaccharide GIP-4 combined with chemotherapy for LLC lung cancer tumor-bearing mice anti-tumor effect experiment and data

[0182] Purpose of the experiment

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

[0184] Experimental materials

[0185] Test sample

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

[0187] Positive control

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

[0189] Experimental animals

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

[0191] Main reagents

[0192] 0.9% sodium chloride injection, batch number: 21071401C, Hunan Kangyuan Pharmaceutical Co., Ltd. product; ALP assay kit, batch number: 201223, purchased from Mikero Bio Co., Ltd. product; 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 from Wako Pure Chemical Industries Co., Ltd. product.

[0193] Main instruments

[0194] S10 type handheld high-speed disperser, Shanghai Xinzhi; AR223CN type electronic balance, Ohaus Instruments (Changzhou) Co., Ltd.; LABOSPECT003 type automatic biochemical analyzer, Hitachi, Ltd., Japan; TDZ5-WS type table type multi-tube automatic balance centrifuge, Hunan Kaidaheng Industrial Development Co., Ltd.; ME2002E type electronic balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.; Digital vernier caliper, Yongkang Zhengfeng Hardware Co., Ltd.; Flow cytometry instrument, BD product; ASP200S type full-automatic tissue dehydrator, ASP300S type full-automatic tissue dehydrator, TP1020 type full-automatic dehydrator, HI1210 type slice spreader, HI1220 type slice baker, RM2235 type paraffin wax slicer, EG1150H+C type tissue embedding machine, AutoStainer XL automatic glass slide stainer + CV5030 automatic cover glass machine, Leica, Germany; BX43 type biological microscope + MD50 type digital imaging system, CX31 type biological microscope, Olympus, Japan.

[0195] Experimental method

[0196] Take the logarithmic growth period Lewis tumor cell strain 1×10 7 / mL 0.2mL was inoculated in the right axillary of 10 pre-purchased normal male C57 mice. The tumor growth was observed to prepare tumor-bearing mice. When the tumor volume of the tumor-bearing mice reached 200mm 3 Above, the tumor tissue of the tumor-bearing mice was taken aseptically to prepare a tissue cell suspension, and the cell culture solution was used to prepare a tumor tissue homogenate at a ratio of 1:1 (V:V). The suspension was inoculated in the right axillary of 60 male C57 mice at a volume of 0.2mL per mouse. When the tumor volume of the tumor-bearing mice reached 100mm 3 Above, the tumor-bearing mice with good growth without rupture were selected and randomly divided into groups according to the tumor volume, namely: model control group, cisplatin group (4mg / kg), Kanglaite soft capsule group (1404mg / kg), cisplatin+Kanglaite soft capsule group (4+1404mg / kg), cisplatin+GLP-4 low-dose group (4+130mg / kg), cisplatin+GLP-4 high-dose group (4+1170mg / kg), 8 mice in each group, and another 8 mice were taken as a normal control group. The cisplatin group of mice was injected intraperitoneally with cisplatin solution, and the rest of the mice in each group was given the corresponding drug solution. The drug volume was 20mL / kg (gargling), 10mL / kg (intraperitoneal injection), 1 time / day, and the continuous administration lasted for 27 days. After the last administration, the blood was collected from the orbit to detect the blood WBC, RBC, liver and kidney function (ALT, AST, BUN, CRE) and CD3 + , CD4 + , CD8 +To determine the content, bone marrow smears were taken for testing, and spleen, thymus, and tumors were weighed and subjected to histopathological examination of the tumors, spleen, stomach, and kidneys.

[0197] Dosage design

[0198] Based on previous research results, Ganoderma lucidum polysaccharide GLP-4 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.

[0199] 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.

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

[0201] Table 1. Trial grouping and dosage design

[0202] detection indicators

[0203] therapeutic indicators

[0204] 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.

[0205] 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.

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

[0207] Attenuation and synergism

[0208] Hematological examination: after the last administration, the blood in the orbit was taken to detect blood routine (WBC, RBC), liver and kidney function index (BUN, CRE).

[0209] Bone marrow examination: after the last administration, bone marrow smear was taken to observe the changes of bone marrow cells in each group of mice.

[0210] Histopathological examination: after the last administration, tumor tissue, spleen, stomach, kidney were taken for HE staining to observe the histopathological changes.

[0211] Blood CD4 + , CD8 + content detection: after the last administration, flow cytometry was used to detect the content of blood lymphocyte typing CD4 + , CD8 + .

[0212] Data processing and statistical analysis

[0213] The effective digits of the data in this experiment were rounded off according to the rounding off method, and statistical analysis was carried out according to the SOP of the center. The statistical software used was SPSS. The measurement data was represented by mean ± standard deviation , and the normality and homogeneity of variance were tested by Leven's test method. If there was no statistical significance (P>0.05), single factor analysis of variance (ANOVA) was used for statistical analysis. If ANOVA had statistical significance (P≤0.05), LSD test (parametric method) was used for comparison analysis. If the variance was not homogeneous (P≤0.05), Kruskal-Wallis test was used. If Kruskal-Wallis test had statistical significance (P≤0.05), Dunnett's Test (non-parametric method) was used for comparison analysis. The statistical results were tested with α=0.05 as the test limit, among which P≤0.05 indicated that there was statistical significance, and P≤0.01 indicated that the difference tested had very significant significance.

[0214] Experimental results

[0215] General clinical observation and animal death

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

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

[0218] Table 2: Number of surviving animals and mortality rate statistics in each group

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

[0220] As shown in Table 3, compared with the normal control group, the body weight of the mice in the model control group was significantly reduced on D4 after administration (P≤0.05). Compared with the model control group, the body weight of the mice in the cisplatin group and the cisplatin combined with Kanleite soft capsule group was significantly reduced on D7-D25 after administration (P≤0.01); the body weight of the mice in the cisplatin combined with GLP-4 low-dose and high-dose groups was significantly reduced on D4-D25 after administration (P≤0.05 or P≤0.01); and the body weight of the mice in the Kanleite soft capsule group was significantly reduced on D10-D19 after administration. Compared with the cisplatin group, the body weight of the mice in the Kanleite soft capsule group was significantly increased on D7-D25 after administration (P≤0.05 or P≤0.01), the body weight of the mice in the cisplatin combined with GLP-4 high-dose group was significantly reduced on D7 after administration (P≤0.05), and the body weight of the mice in the cisplatin combined with GLP-4 low-dose group was significantly reduced on D7-D13 after administration (P≤0.05 or P≤0.01). Compared with the Kanleite soft capsule group, the body weight of the mice in the cisplatin combined with Kanleite soft capsule group and the GLP-4 low-dose and high-dose groups was significantly reduced on D7-D25 after administration (P≤0.05 or P≤0.01). Compared with the cisplatin + Kanleite soft capsule group, the body weight of the mice in the cisplatin combined with GLP-4 low-dose group was significantly reduced on D7-D13 after administration (P≤0.05 or P≤0.01), and there was no statistical significance in the other groups.

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

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

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

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

[0225] 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 GLP-4 low-dose and high-dose groups was significantly reduced on D13-D25 (P≤0.05 or P≤0.01). Compared with the cisplatin + Kanglaite soft capsule group, the tumor volume of mice in the cisplatin combined with GLP-4 low-dose group was significantly reduced on D4-D13 (P≤0.05 or P≤0.01), and there was no statistical significance in the other groups.

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

[0227] Note: compared with the model control group, *P≤0.05, **P≤0.01; compared with the cisplatin group, # P≤0.05, ## P≤0.01; compared with 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.

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

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

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

[0231] As shown in Table 6, the T / C of mice in the cisplatin + GLP-4 low-dose group on D4-D25 were 42.0%, 27.3%, 3.0%, 0.0%, 0.0%, 0.8%, 1.8%, 2.1%, all of which were ≤60.0%; the T / C of mice in the cisplatin + GLP-4 high-dose group on D4-D25 were 57.1%, 27.3%, 3.0%, 0.0%, 0.0%, 2.7%, 4.0%, 4.1%, all of which were ≤60.0%; the T / C of mice in the cisplatin group on D7-D10 and D16-D25 were 45.5%, 27.3%, 28.2%, 36.9%, 30.9%, 23.6%, all of which were ≤60.0%; the T / C of mice in the cisplatin + Kanglaite soft capsule group on D13-D25 were 7.3%, 3.3%, 15.6%, 7.3%, 15.6%, all of which were ≤60.0%.

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

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

[0234] 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), while 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 low-dose GLP-4 group, and the Kanglaite soft capsule group were significantly decreased (P≤0.05 or P≤0.01), while the spleen and tumor coefficients of mice in the cisplatin combined with high-dose GLP-4 group were significantly decreased (P≤0.01). Compared with the cisplatin group, the spleen and tumor coefficients of mice in the Kanglaite soft capsule group were significantly increased (P≤0.01). Compared with the Kanglaite soft capsule group, the spleen and tumor coefficients of mice in the cisplatin combined with high-dose GLP-4 group were significantly decreased (P≤0.01), while the spleen, thymus, and tumor coefficients of mice in the cisplatin combined with low-dose GLP-4 group were significantly decreased (P≤0.05 or 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.

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

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

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

[0238] In Figure 22, the following labels are used for comparison: 1: Model control group; 2: Cisplatin group; 3: Kanglaite soft capsule group; 4: Cisplatin + Kanglaite soft capsule group; 7: Cisplatin + GLP-4 low-dose group; 8: Cisplatin + GLP-4 high-dose group.

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

[0240] As shown in Table 8, compared with the normal control group, the model control group mice blood WBC, ALT, AST, BUN content were significantly increased (P≤0.05 or P≤0.01), RBC was significantly reduced (P≤0.05). Compared with the model control group, cisplatin combined with GLP-4 low, high dose group of mice blood WBC, AST were significantly reduced (P≤0.05 or P≤0.01), cisplatin group, cisplatin combined with Kanglaite soft capsule group WBC were significantly reduced (P≤0.05). Compared with cisplatin group, Kanglaite soft capsule group of mice blood BUN was significantly increased (P≤0.05), cisplatin combined with Kanglaite soft capsule group of mice blood RBC was significantly reduced (P≤0.05). Compared with Kanglaite soft capsule group, cisplatin combined with Kanglaite soft capsule group of mice blood WBC was significantly reduced (P≤0.05). Compared with cisplatin + Kanglaite soft capsule group, each group was not statistically significant.

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

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

[0243] Effects of Ganoderma polysaccharide GLP-4 combined with chemotherapy on CD3 + / CD4 + , CD3 + / CD8 + of Lewis lung cancer tumor-bearing mice

[0244] As shown in Table 9, compared with the normal control group, the model control group mice blood CD3 + / CD8 + was significantly increased (P≤0.05). Compared with the model control group, cisplatin combined with GLP-4 low dose group of mice blood CD3 + / CD4 + was significantly reduced (P≤0.05). Compared with the cisplatin group, cisplatin combined with GLP-4 low dose group of mice blood CD3 + / CD4 + , CD3 + / CD8 +Significantly reduced (P≤0.05); the blood CD3 + / CD8 + Significantly reduced (P≤0.05). Compared with the Kanglaite soft capsule group, the blood CD3 + / CD4 + Significantly reduced (P≤0.05), the blood CD3 + / CD8 + Significantly increased (P≤0.05). Compared with the cisplatin + Kanglaite soft capsule group, the blood CD3 + / CD4 + , CD3 + / CD8 + Significantly reduced (P≤0.05), and the rest of the administration groups had no statistical significance.

[0245] Table 9 Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on CD3 + / CD4 + , CD3 + / CD8 + content of Lewis lung cancer-bearing mice

[0246] Note: Compared with the normal control group, + P≤0.05; compared with the model control group, *P≤0.05; compared with the cisplatin group, # P≤0.05; compared with 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.

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

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

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

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

[0251] Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on tumor, spleen, kidney and stomach histopathology of Lewis cell-bearing mice

[0252] As shown in Figure 23, the model control group had a large number of tumor cells around the tumor, the cells were arranged closely, the cell volume was large, the nucleus-cytoplasm ratio was large, and mitotic phase was common. In the cisplatin group, a large number of necrosis and hemorrhage were observed in the tumor center, and the number of tumor cells was rich. In the cisplatin + Kanglete soft capsule group and the cisplatin + GLP-4 low dose group, necrosis was observed in the center of the tumor tissue, and the number of tumor cells around the tumor was reduced. In the cisplatin + GLP-4 high dose group, vacuoles were observed in the tumor tissue.

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

[0254] As shown in Figure 26, the spleen red pulp hematopoiesis of the model control group was obvious, and the cell types were diverse. No obvious abnormalities were found in the spleen of the cisplatin + Kanglete soft capsule group, and different degrees of red pulp hematopoiesis and diverse cell types were found in the spleen of the other groups.

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

[0256] Conclusion

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

[0258] Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy for H22 hepatocarcinoma tumor-bearing mice anti-tumor effect experiment and data

[0259] Purpose of the experiment

[0260] C57 mice were inoculated with H22 hepatocarcinoma tumor homogenate in the right armpit to prepare tumor-bearing mice, and the effect of Ganoderma lucidum polysaccharide GLP-4 on hepatocarcinoma tumor-bearing mice was studied to provide experimental basis for clinical research.

[0261] Experimental materials

[0262] Test product

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

[0264] Positive control

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

[0266] Experimental animals

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

[0268] Main reagents

[0269] PBS buffer prepared by Shenzhen Olimed Oncology Medical Technology Co., Ltd.; fetal bovine serum, product of Zhejiang Tianhang Biological Technology Co., Ltd.; 1640 culture medium, product of Gibco Company; 0.25% trypsin, product of Gibco Company.

[0270] Main instruments

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

[0272] Experimental method

[0273] H22 hepatocarcinoma solid tumor homogenate suspension was injected subcutaneously into the armpit of 60 healthy male C57 mice to make a solid tumor model. When the average tumor volume of all mice was 180mm 3 , the mice were randomly divided according to the tumor volume, and the corresponding drugs or drug vehicles were given by gavage or intraperitoneal injection, respectively, for 27 consecutive days. The tumor length and short diameter were measured every 3 days, and the tumor volume was calculated. The mouse body weight was measured every 3 days. At the end of the experiment, the mouse tumors, spleen and thymus were dissected and weighed, and the tumor index, spleen index and thymus index were calculated.

[0274] Dose design

[0275] According to the previous experimental results, the design of Ganoderma lucidum polysaccharide GLP-4, low dose: 50mg / kg, high dose: 150mg / kg, according to Table 11, the corresponding drugs were given respectively.

[0276] The dose design of cisplatin is based on: according to the clinical dose of cisplatin, each person per day does not exceed 100mg / m 2 , and the tolerance of mice to cisplatin, 3mg / kg dose was selected as the dose.

[0277] Table 11 Test grouping and dose design

[0278] Detection index

[0279] Efficacy index

[0280] Relative tumor inhibition rate

[0281] Relative tumor inhibition rate (%) = (1-T RTV / C RTV ) x 100%. Wherein, TRTV Relative tumor volume of experimental group, C RTV Relative tumor volume of model control group. Relative tumor volume RTV = V t V0, V t Tumor volume of mice on the tth day of administration, V0is the tumor volume of mice at the time of grouping. Evaluation criteria: relative tumor inhibition rate ≥ 40%, and statistical analysis P < 0.05 is effective inhibition level.

[0282] Tumor growth inhibition rate

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

[0284] Spleen, thymus organ coefficient: after the last administration, the spleen, thymus, tumor was weighed and the organ coefficient was calculated.

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

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

[0287] Data processing and statistical analysis

[0288] Statistical analysis was performed using SPSS17.0, and the level of statistical significance was set as P≤0.05, and the measurement data was expressed as mean ± standard deviation , Leven's test method was used to test normality and homogeneity of variance, if normality and homogeneity of variance were met (P>0.05), single factor analysis of variance (ANOVA) and LSD test were used for statistical analysis; if normality and homogeneity of variance were not met (P<0.05), Kruskal-Wallis test was used, if Kruskal-Wallis test had statistical significance (P<0.05), Dunnett's Test (non-parametric method) was used for comparison analysis. Statistical difference and biological significance were considered in evaluation

[0289] Experimental results

[0290] Animal death

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

[0292] Table 12 Survival of animals in each group and mortality statistics

[0293] Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on body weight of H22 hepatoma-bearing mice

[0294] As shown in Table 13, compared with the normal group, the model control group had a significant increase in mouse weight from D0 to D27; the cisplatin group had a significant increase in mouse weight at D0 and D3, and a significant decrease in mouse weight from D15 to D27; the cisplatin+GLP-4 low-dose group had a significant decrease in mouse weight from D12 to D27; and the cisplatin+GLP-4 high-dose group had a significant decrease in mouse weight from D9 to D27.

[0295] Compared with the model control group, the cisplatin group had a significant decrease in mouse weight from D6 to D27, and the cisplatin+GLP-4 low-dose and high-dose groups had a significant decrease in mouse weight from D3 to D27.

[0296] Compared with the cisplatin group, the cisplatin+GLP-4 high-dose group had a significant decrease in mouse weight at D15 and D24.

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

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

[0299] Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on tumor volume of H22 hepatoma-bearing mice

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

[0301] Compared with the cisplatin group, the cisplatin+GLP-4 low-dose group had a significant decrease in tumor volume at D12 to D18 and D27.

[0302] Table 14 Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on tumor volume of H22 hepatoma-bearing mice

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

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

[0305] Compared with the cisplatin group, the relative tumor inhibition rates of the low-dose cisplatin + GLP-4 group were 44.4%, 60.9%, 55.6%, and 63.3% on days 12–18 and 27, respectively (P<0.05), while the relative tumor inhibition rates of the high-dose cisplatin + GLP-4 group were 41.6% and 42.3% on days 15 and 27, respectively.

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

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

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

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

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

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

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

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

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

[0315] As shown in Figures 31-34, they are pictures of tumor-bearing mice, and the corresponding groups are: Figure 31: model control group, Figure 32: cisplatin group, Figure 33: cisplatin + GLP-4 low-dose group, and Figure 34: cisplatin + GLP-4 high-dose group

[0316] Conclusion

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

[0318] Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy for H22 orthotopic hepatoma mice and data

[0319] Purpose of the experiment

[0320] In this experiment, C57 mice were inoculated with H22 hepatoma cells in the right axillary to prepare orthotopic tumor mice, and the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 orthotopic hepatoma mice was studied to provide experimental basis for clinical research.

[0321] Experimental materials

[0322] Test sample

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

[0324] Reference substance

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

[0326] Experimental animals

[0327] SPF level male C57 mice 65, weighing 12-15 g, provided by Hunan Slike Jingda Experimental Animal Co., Ltd., experimental animal production license number: SCXK(Xiang)2019-0004, fed in the barrier environment animal laboratory D area of Hunan Puremax Pharmaceutical Research Center Co., Ltd., experimental animal use license number: SYXK(Xiang)2020-0015.

[0328] Main reagents

[0329] 0.9% sodium chloride injection, batch number: 21071401C, Hunan Kangyuan Pharmaceutical Co., Ltd. product; 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 Japan Wako Pure Chemical Industries Co., Ltd.

[0330] Main instruments

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

[0332] Experimental method

[0333] The concentration of Luc labeled fluorescence was 1×10 7Hepatocellular carcinoma (H22) cells at a density of 10 cells / mL were first inoculated into the peritoneal cavity of 8 male C57 mice. Once ascites developed in the mice, the ascites fluid was aseptically aspirated, washed with HBSS buffer, centrifuged, and the supernatant discarded. The ascites fluid was stained with trypan blue and the cells were counted under a microscope. The cell count was then adjusted to 1 × 10⁶ cells / mL with HBSS buffer. 13 Fifty-one male C57 mice were injected with 10 μL / mouse into the right hepatic region to create orthotopic tumor-bearing mice. One week later, tumor formation in the liver was assessed using a small animal in vivo imaging system. Mice were randomly assigned to four groups based on tumor size: a model control group, a cisplatin group (4 mg / kg), a cisplatin-containing soft capsule group (1404 mg / kg), a cisplatin + cisplatin soft capsule group (4 + 1404 mg / kg), a cisplatin + GLP-4 low-dose group (4 + 130 mg / kg), and a cisplatin + GLP-4 high-dose group (4 + 1170 mg / kg), with six mice in each group. Six mice served as a normal control group. The normal control and model control groups were administered pure water by gavage. Mice in the chemotherapy groups received intraperitoneal injections of cisplatin. The remaining groups received the corresponding drug solutions at doses of 20 mL / kg (gavage) or 10 mL / kg (intraperitoneal injection), once daily for 14 consecutive days. After the last dose, blood samples were drawn from the orbital rim to measure blood WBC, RBC, liver and kidney function indicators (ALT, AST, BUN, CRE), and CD3. + / CD4 + CD3 + / CD8 + The organ coefficients were calculated by weighing the spleen, thymus, and tumor.

[0334] Dosage design

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

[0336] The proposed clinical dosage of Kanglaite soft capsules is 0.45g / capsule, 6 capsules / 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.

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

[0338] Table 18 Trial grouping and dosage design

[0339] detection indicators

[0340] Therapeutic index

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

[0342] Tumor volume detection: The change of tumor volume in each group was detected every week by small animal live imaging technology.

[0343] Hematological detection: Blood routine (WBC, RBC), biochemical (liver and kidney function) index detection after the last administration.

[0344] Immune organs: Thymus, spleen, tumor, liver were weighed and organ coefficients were calculated. Organ coefficient (%) = organ weight / body weight after fasting x 100%

[0345] CD4 + , CD8 + content detection: After the last administration, flow cytometry was used to detect the blood lymphocyte typing CD3 + / CD4 + , CD3 + / CD8 + content.

[0346] Data processing and statistical analysis

[0347] The effective number of test data was rounded off according to the rounding method, and statistical analysis was carried out according to the central SOP, and the statistical software used was SPSS. Measurement data was represented by mean ± standard deviation , normality and homogeneity of variance were tested by Leven's test method. If there was no statistical significance (P>0.05), single factor analysis of variance (ANOVA) was used for statistical analysis. If ANOVA had statistical significance (P≤0.05), LSD test (parametric method) was used for comparative analysis. If the variance was not uniform (P≤0.05), Kruskal-Wallis test was used. If Kruskal-Wallis test had statistical significance (P≤0.05), Dunnett's Test (non-parametric method) was used for comparative analysis. The statistical results were tested with α=0.05 as the test limit, P≤0.05 indicated statistical significance, and P≤0.01 indicated that the difference tested had very significant significance.

[0348] Experimental results

[0349] General clinical observation and animal death

[0350] As shown in Table 19, the mice were active before administration, and their movements and gaits were normal; after administration, the mice were less active, and their diet and water intake were affected after tumor growth, and their body weight increased less. The model control group 2M01 died on D12; the cisplatin groups 3M01 / 3M05 and 3M02 died on D9 and D10, respectively; the Kanglete soft capsule group 4M01 died on D9; the cisplatin+Kanglete soft capsule groups 5M01 and 5M06 died on D8 and D11, respectively; and the cisplatin+GLP-4 low-dose group 8M02 died on D8.

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

[0352] Table 19: Number of surviving animals and mortality rate in each group

[0353] Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on body weight of H22 orthotopic liver cancer mice

[0354] As shown in Table 20, compared with the normal control group, the body weight of the mice in the model control group was significantly reduced at W2 weeks after administration (P≤0.01); compared with the model control group, the body weight of the mice in the cisplatin+GLP-4 low-dose group and the cisplatin+GLP-4 high-dose group was significantly reduced at W1 and W2 weeks after administration (P≤0.05 or P≤0.01), and the body weight of the mice in the cisplatin group and the cisplatin+Kanglete soft capsule group was significantly reduced at W1 and W2 weeks after administration (P≤0.05 or P≤0.01). Compared with the cisplatin group, the body weight of the mice in the Kanglete soft capsule group was significantly increased at W2 weeks after administration (P≤0.05 or P≤0.01). Compared with the Kanglete soft capsule group, the body weight of the mice in the cisplatin+GLP-4 low-dose group and the cisplatin+GLP-4 high-dose group was significantly reduced at W1 and W2 weeks after administration (P≤0.05 or P≤0.01). Compared with the cisplatin+Kanglete soft capsule group, the body weight of the mice in the cisplatin+GLP-4 high-dose group was significantly reduced at W2 weeks after administration (P≤0.05 or P≤0.01).

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

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

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

[0358] As shown in Figures 35, 36 and Table 21, compared with the normal control group, the tumor of the model control group mice was significantly increased (P < 0.01); compared with the model control group, the tumor of the Kanglaishu soft capsule group mice administered at W1 and W2 was significantly reduced (P < 0.05 or P < 0.01); the tumor of the cisplatin combined with GLP-4 low and high dose groups, cisplatin group, cisplatin combined with Kanglaishu soft capsule group mice administered at W2 was significantly reduced (P < 0.05 or P < 0.01). Compared with the cisplatin group, there was no significant difference in each administration group. Compared with the Kanglaishu soft capsule group, the tumor of the cisplatin combined with GLP-4 high dose group and Kanglaishu soft capsule group mice administered at W1 was significantly increased (P < 0.05 or P < 0.01). Compared with the cisplatin + Kanglaishu soft capsule group, there was no statistical difference in each group.

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

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

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

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

[0363] Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on H22 orthotopic liver cancer in mice

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

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

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

[0367] Effect of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on blood biochemical indicators of H22 orthotopic liver cancer mice

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

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

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

[0371] As shown in Table 24, compared with the normal control group, the model control group mice had a rising trend in blood CD3+ / CD4+and CD3+ / CD8+, but there was no statistical significance. Compared with the model control group, the cisplatin combined with GLP-4 high-dose group mice had significantly increased blood CD3 + / CD8 + (P≤0.05); the cisplatin combined with GLP-4 high-dose group mice had significantly increased blood CD3 + / CD4 + and CD3 + / CD8 + (P≤0.05). Compared with the cisplatin group, the cisplatin combined with GLP-4 low-dose and high-dose group mice had significantly increased blood CD3 + / CD4 + and CD3 + / CD8 + (P≤0.05). Compared with the Kanglaite soft capsule group, the cisplatin combined with GLP-4 low-dose group mice had significantly increased blood CD3 + / CD4 + and CD3 + / CD8 + (P≤0.05), and the cisplatin combined with GLP-4 high-dose group mice had significantly increased blood CD3 + / CD4 + (P≤0.05). Compared with the cisplatin + Kanglaite soft capsule group, the cisplatin combined with GLP-4 low-dose and high-dose group mice had significantly increased blood CD3 + / CD4 + (P≤0.05).

[0372] Table 24 Effects of Ganoderma lucidum polysaccharide GLP-4 combined with chemotherapy on blood CD3 + / CD4 + and CD3 + / CD8+ Effects of the combination of GLP-4 and cisplatin

[0373] Note: 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; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05.

[0374] As shown in the results of FIGS. 37-40, a large number of liver cancer cell infiltrations and necrosis occurred in the liver of the model control group mice, the number of liver sinusoidal cells increased, and inflammatory cell infiltrations occurred; extramedullary hematopoiesis in the spleen increased significantly, the red pulp was diffuse, liver cancer cell infiltrations occurred in the stomach, lesions occurred in the serosa, and the gastric mucosa was widely atrophied. After administration of Ganoderma lucidum polysaccharide GLP-4 combined with cisplatin, the degree of liver cancer cell infiltration and necrosis in the liver of each group decreased, extramedullary hematopoiesis in the spleen increased, and no obvious lesions were found in the stomach and kidney.

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

[0376] Conclusion

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

[0378] Discussion and summary

[0379] Liver cancer is a malignant tumor with a very high mortality rate. At present, surgical resection combined with chemotherapy and interventional therapy, radiotherapy and other treatments can only delay symptoms and it is very difficult to completely cure. Liver cancer is not sensitive to chemotherapy, especially for patients with advanced liver cancer. To date, there is no reliable evidence to prove that systemic chemotherapy can improve the overall survival of patients with advanced liver cancer.

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

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

[0382] Experiment purpose:

[0383] Experiment purpose:

[0384] To explore the co-localization of GLP-4 and RAW264.7 macrophage lysosomes and further elucidate the mechanism of drug efficacy, providing experimental basis for clinical research.

[0385] Experimental materials

[0386] Test sample

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

[0388] Cell line

[0389] RAW264.7 macrophage cell line, purchased from Shanghai Institute of Life Sciences, Chinese Academy of Sciences.

[0390] Main reagents

[0391] PBS, prepared by Shenzhen Olimed Oncology Medical Technology Co., Ltd.; DMEM medium, product of Gibco Company; Cell Navigator lysosome staining kit, product of Beijing Solaybao Technology Co., Ltd.; DAPI dye, product of Tianjin Baima Technology Co., Ltd.; HBSS buffer, product of Gibco Company.

[0392] Main instruments

[0393] 20 μL, 200 μL, 1 mL pipette, Mettler-Toledo Limited product; Luna-II cell counter, Nanjing Hengqiao Instrument Co., Ltd. product; laser confocal microscope, Leica product.

[0394] Experimental method:

[0395] RAW264.7 macrophages were cultured in DMEM medium containing 10% serum, 37°C, 5% CO2 incubator to logarithmic growth phase. Add 150,000 cells in six-hole plate, culture 1-2 days, when the cell density reaches more than 50%, add fluorescent labeled GLP-4 incubate for 24 hours, then add lysosome staining agent and DAPI dye, take pictures under 488nm, 550nm and ultraviolet light by laser confocal microscope.

[0396] Test results

[0397] As shown in the results of Figures 41-45, after incubation of Ganoderma lucidum polysaccharide GLP-4 with RAW264.7 macrophages for 24 hours, most of the cells contained drugs inside, indicating that GLP-4 can be phagocytosed by macrophages; the pictures of co-localization of fluorescent labeled GLP-4 and lysosomes showed that the drugs entered the macrophages through the endocytosis pathway of lysosomes; in Figure 44, the dead cells labeled by DAPI dye contained fluorescent labeled GLP-4, but there was no lysosome in the cells, indicating that the lysosomes of macrophages cannot fully digest the drugs, so GLP-4 can be presented to other immune cells by macrophages, which can successfully activate the immune system and achieve the effect of enhancing immunity.

[0398] As shown in Figure 41, it is the confocal picture of RAW264.7 macrophage blank control group under 40 times lens. A: is the merged picture of bright field picture and DAPI staining picture; B: is the picture under FITC fluorescence channel; C: is the picture of lysosome labeled by lysosome fluorescent dye; D: is the picture after merging C picture and bright field picture.

[0399] As shown in Figure 42, it is the confocal picture of 0.25 mg / mL GLP-4 incubated with RAW264.7 macrophages for 24 hours under 40 times lens. A: is the picture after merging bright field picture and DAPI staining picture; B: is the picture of FITC labeled GLP-4; C: is the picture of lysosome labeled by lysosome fluorescent dye; D: is the picture after merging B picture and C picture, the arrow shows the picture of co-localization of GLP-4 and lysosome.

[0400] As shown in Figure 43, it is the confocal picture of 0.25 mg / mL GLP-4 after incubation with RAW264.7 macrophages for 24 hours under 63 times oil lens. A: is the picture of the combination of bright field picture and DAPI staining picture; B: is the picture of FITC labeled GLP-4; C: is the picture of lysosome labeled by lysosome fluorescent dye; D: is the picture of the combination of B and C, and the arrow shows the picture of GLP-4 and lysosome co-localization.

[0401] As shown in Figure 44, it is the confocal picture of 0.5 mg / mL GLP-4 after incubation with RAW264.7 macrophages for 24 hours under 40 times lens. A: is the picture of the combination of bright field picture and DAPI staining picture; B: is the picture of FITC labeled GLP-4; C: is the picture of lysosome labeled by lysosome fluorescent dye; D: is the picture of the combination of B and C, and the arrow shows the picture of GLP-4 and lysosome co-localization.

[0402] As shown in Figure 45, it is the confocal picture of 0.5 mg / mL GLP-4 after incubation with RAW264.7 macrophages for 24 hours under 63 times oil lens. A: is the picture of the combination of bright field picture and DAPI staining picture; B: is the picture of FITC labeled GLP-4; C: is the picture of lysosome labeled by lysosome fluorescent dye; D: is the picture of the combination of B and C, and the arrow shows the picture of GLP-4 and lysosome co-localization.

[0403] Conclusion

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

[0405] Research experiment and data of killing effect of ganoderma lucidum polysaccharide GLP-4 combined with cisplatin on H22 hepatoma cells

[0406] Experimental purpose:

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

[0408] Experimental materials

[0409] Test sample

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

[0411] Positive control

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

[0413] Cell lines

[0414] H22 hepatoma cell line, purchased from Shanghai Institute of Life Sciences, Chinese Academy of Sciences.

[0415] Main reagents

[0416] PBS, prepared by Shenzhen Olimed Tumor Medical Technology Co., Ltd.; 0.25% trypsin, product of Gibco Company; 1640 culture medium, product of Gibco Company.

[0417] Main instruments

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

[0419] Experimental method:

[0420] H22 cells were cultured in 1640 culture medium containing 10% serum in a 37℃, 5% CO2 incubator to the logarithmic growth phase. The cells in the culture bottle were discarded, washed with PBS, and 0.125% trypsin was added for digestion. After digestion, the cells were transferred to an EP tube and counted using a cell counter. After counting, 4,000 cells per well were added to a 12-well plate and cultured in a 37℃, 5% CO2 incubator. After 24 hours, the cells were adhered, and drugs were added. After 48 hours, the cells were digested with trypsin, washed with PBS, and counted. According to the number of cells, the corresponding volume of staining solution was added, incubated in the incubator for 30 minutes, and then placed in an ice box to terminate staining and maintain cell activity. The cells were photographed using a laser confocal microscope.

[0421] Dose design

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

[0423] Test results

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

[0425] Table 25 Effect of ganoderma lucidum polysaccharide GLP-4 combined with cisplatin on H22 cell necrosis and apoptosis

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

[0427] Conclusion

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

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

[0430] Experimental purpose

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

[0432] Experimental materials

[0433] Test sample

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

[0435] Positive control

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

[0437] Experimental animals

[0438] 65 SPF female BALB / c mice, weighing 14-16 g, were provided by Guangdong Medical Laboratory Animal Center, experimental animal production license number: SCXK (Yue) 2022-0002, experimental animal quality certificate number: 44007200100339.

[0439] Main reagents

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

[0441] Main instruments

[0442] Vernier caliper, Shanghai Tool Factory Co., Ltd. product; I-2000 type balance, Dongguan City Nan Cheng Chang Xie Electronic Products Factory; Ophthalmic scissors, ophthalmic forceps, Shanghai Jinzhong Medical Instrument Co., Ltd. product; CCL-170B-8 carbon dioxide incubator, Singapore ESCO company product; Luna-II cell counter, Nanjing Hengqiao Instrument Co., Ltd. product.

[0443] Experimental method

[0444] 4T1 breast cancer cell suspension was injected into the left shoulder of 55 healthy female BALB / c mice subcutaneously to make a solid tumor model. When the average tumor volume of all mice was about 240mm3, the mice were randomly divided into groups according to the tumor volume, and the corresponding drugs or drug vehicles were given by gavage or intraperitoneal injection, respectively, for 25 consecutive days. The tumor length and short diameter were measured every 3 days, and the tumor volume was calculated. The mice body weight was measured every 3 days. At the end of the experiment, the mouse tumor, spleen and thymus were weighed, and the tumor index, spleen index and thymus index were calculated.

[0445] Dose design

[0446] According to the previous experimental results, the design of Ganoderma lucidum polysaccharide GLP-4, low dose: 130mg / kg, high dose: 1170mg / kg, according to table 26 respectively given corresponding drug.

[0447] The dose design of cisplatin administration is based on: according to the clinical dose of cisplatin, each person per day is not more than 100mg / m 2 and the tolerance of mice to cisplatin, 4mg / kg dose is selected as the administration dose.

[0448] Table 26 test grouping and dose design

[0449] Detection index

[0450] Therapeutic index

[0451] Relative tumor inhibition rate

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

[0453] Tumor growth inhibition rate

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

[0455] Spleen, thymus organ coefficient: after the last administration, the spleen, thymus, tumor was weighed and the organ coefficient was calculated.

[0456] Data processing and statistical analysis

[0457] Statistical analysis was performed using SPSS17.0, and the level of statistical significance was set as P≤0.05. Measurement data was expressed as mean ± standard deviation (x±s), and normality and homogeneity of variance were tested by Leven's test method. If normality and homogeneity of variance were met (P>0.05), single factor analysis of variance (ANOVA) and LSD test were used for statistical analysis; if normality and homogeneity of variance were not met (P<0.05), Kruskal-Wallis test was used, and if Kruskal-Wallis test had statistical significance (P<0.05), Dunnett's Test (non-parametric method) was used for comparison analysis. Statistical differences and biological significance were considered in evaluation

[0458] Experimental results

[0459] Animal death

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

[0461] Table 27 Survival of animals in each group and mortality statistics

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0483] As shown in Figures 51-54, they are pictures of tumor-bearing mice, and the corresponding groups are: Figure 51: model control group, Figure 52: cisplatin group, Figure 53: cisplatin + GLP-4 low dose group, Figure 54: cisplatin + GLP-4 high dose group

[0484] Conclusion

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

[0486] Pharmacodynamic experiment and data of bleomycin-induced pulmonary fibrosis animal model

[0487] Experimental purpose

[0488] In this study, a bleomycin-sensitized mouse pulmonary fibrosis model was used to investigate the therapeutic effect of Ganoderma lucidum polysaccharide GLP-4 on pulmonary fibrosis, and to provide experimental evidence for its clinical research.

[0489] Experimental materials

[0490] Test product

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

[0492] Positive control

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

[0494] Experimental animals

[0495] SPF C57BL / 6J mice 50, body weight 21.1-25.0 g, purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd., experimental animal production license number: SCXK (Yue) 2020-0063. Experimental animal quality certificate number: No: 44829700008922.

[0496] Main reagents

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

[0498] Main instruments

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

[0500] Experimental method

[0501] Take 50 SPF male C57BL / 6J mice, randomly group according to body weight, that is, normal control group, model control group, positive control group, GLP-4 low dose group, GLP-4 high dose group, select 10 animals as normal control group without modeling, the rest of the animals are modeled, 10 in each group. Modeling through tracheal administration of bleomycin to mice, 5 mg / kg, 0.1 mL per mouse, 1 time of administration. On the 7th day of modeling, the test drug, positive drug or vehicle was given by gavage, 10 mL / kg, once a day, for 21 consecutive days. During the test period, the diet, activity, respiration and response ability of mice in each group were observed every day; before administration, about 24 h after the last administration, the lung function of mice was detected; after lung function detection, the animals were euthanized and pathological samples were taken.

[0502] Dose design

[0503] The clinical dosage of Ganoderma lucidum polysaccharide GLP-4 is 1.0 g per day. According to the body surface area conversion coefficient of 0.0026 between human and mice, the clinical equivalent dose of mice is about 187 mg / kg. The low (approximately clinical equivalent dose) and high doses of GLP-4 are designed as 200 mg / kg and 600 mg / kg, respectively.

[0504] The positive drug pirfenidone is calculated as a stable daily dose of 1200 mg for adults. The clinical equivalent dose of mice is 225 mg / kg, which is set as the dose of the positive control group. As shown in Table 33.

[0505] Table 33: Dose design and administration amount table

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

[0507] Detection index

[0508] General behavior observation: During the test period, the diet, activity, respiration, and response ability of animals in each group were observed every day.

[0509] Pulmonary function: Before administration and about 24 h after the last administration, the pulmonary function of mice was detected by EMKA WBP animal pulmonary function detection system, which can include the inspiratory (expiratory) duration, maximum inspiratory (expiratory) volume, tidal volume, expiratory volume, relaxation time, minute ventilation, respiratory rate, inspiratory (expiratory) end gap, bronchial constriction parameter, 50% expiratory flow, alveolar ventilation, and respiratory frequency of mice.

[0510] Lung histopathology: After the pulmonary function test, the mice were sacrificed, the abdomen was opened, and the whole body was perfused with normal saline through the portal vein. The chest was opened, and the trachea was ligated after perfusion fixation with 10% formaldehyde solution for 5 min. The lung was taken out and fixed with formalin solution for 48 hours for HE staining to observe the lesion.

[0511] Data processing and statistical analysis

[0512] The experimental measurement data were summarized by Excel and represented by mean ± standard deviation. The statistical software SPSS 28.0 was used for analysis. The variance homogeneity test was performed by one-way ANOVA. When the variance was homogeneous (P≥0.05), the LSD test was used for comparison between groups. When the variance was not homogeneous (P<0.05), the Dunnett’s T3 test was used for comparison between groups. P<0.05 was considered statistically significant.

[0513] Experimental results

[0514] General behavior observation

[0515] During the experiment, compared with the normal control group, the model control group mice had poor appetite, reduced activity, rapid breathing, reduced reaction ability, and dull fur. Compared with the model control group, the positive control group, GLP-4 low-dose group, and GLP-4 high-dose group had significant improvements in appetite, activity, breathing, reaction ability, and fur.

[0516] Pulmonary function test

[0517] Before administration (1 week after modeling), the pulmonary function was as shown in Tables 34 and 35. Compared with the normal control group, the model control group mice had significantly increased inspiratory duration (Ti), maximum inspiratory volume (PIF), relaxation time (RT), end inspiratory pause (EIP), and respiratory rate (n) (p<0.01 or 0.05), and significantly decreased minute ventilation (MV) and bronchial constriction parameter (Penh) (p<0.01). The positive drug control group and GLP-4 low and high dose groups had consistent results with the model control group. The above results suggest that the animals in the modeling groups have abnormal lung function, and the model is successful. Compared with the model control group, the positive drug control group had statistically significant differences in maximum expiratory flow (PEF), MV, 50% expiratory flow (EF50), and n, which should be caused by individual differences and detection errors and should have no clinical significance.

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

[0519] Table 34 Pulmonary function test results Table 1

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

[0521] Table 35 Results of pulmonary function test Table 2

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

[0523] Table 36 Results of pulmonary function test Table 3

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

[0525] Table 37 Results of pulmonary function test Table 4

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

[0527] Pathological examination

[0528] The pathological test results are shown in Table 38. Compared with the normal control group, the lung pathological score of the model control group was significantly increased (p<0.01); compared with the model control group, the lung pathological score of the GLP-4 low dose group and the GLP-4 high dose group was significantly reduced (p<0.01 or 0.05).

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

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

[0531] As shown in Figures 55-59, the alveolar structure of the normal control group mice was normal, no inflammatory cell infiltration was observed, and no fibrous tissue hyperplasia was observed. The lung tissue of the model control group mice was damaged (40-60%), inflammatory cells infiltrated the lung tissue (40-60%), and fibrous tissue hyperplasia was observed around the blood vessels and in the lung tissue (40-60%). The lung tissue of the positive control group mice was damaged (1-20%), inflammatory cells infiltrated the lung tissue (1-20%), and no fibrous tissue hyperplasia was observed. The alveolar structure of the GLP-4 low dose group mice was normal, inflammatory cells infiltrated the lung tissue (1-20%), and no fibrous tissue hyperplasia was observed around the blood vessels and in the lung tissue. The lung tissue of the GLP-4 high dose group mice was damaged (1-20%), inflammatory cells infiltrated the lung tissue (1-20%), and no fibrous tissue hyperplasia was observed around the blood vessels and in the lung tissue.

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

[0533] Conclusion

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

[0535] In conclusion, under the experimental conditions, Ganoderma lucidum polysaccharide GLP-4 can improve the general behavior, lung function and lung tissue lesions of the asthma model mice, and has a therapeutic effect on pulmonary fibrosis.

[0536] The above merely describes preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

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

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

3. A method for extracting Ganoderma lucidum polysaccharide GLP-4, characterized in that, The method comprises the following steps: S1, after the Ganoderma lucidum is dried and dusted, it is crushed to form Ganoderma lucidum powder; S2, the crushed Ganoderma lucidum is placed in a sealed container and mixed with water and heated, and the Ganoderma lucidum powder and water are fully combined into a medicinal juice solution under high temperature and high pressure; S3, the medicinal juice solution is separated by centrifugation to obtain a concentrated solution of medicinal ingredients and medicinal residues; S4, the separated medicinal residues are mixed with a NaOH solution according to a predetermined ratio, soaked, and then neutralized by adding HCl; S5, the mixed solution is concentrated and desalted by membrane concentration technology to remove the NaCl component in the mixed solution to obtain a concentrated solution of medicinal ingredients; S6, the concentrated solution containing medicinal ingredients is freeze-dried to a certain concentration and separated by column chromatography multiple times to obtain Ganoderma lucidum polysaccharide GLP-4 with medicinal ingredients.

4. The method of claim 3, wherein the extraction of Ganoderma lucidum polysaccharide GLP-4 is characterized by, In step S2, the Ganoderma lucidum powder and water in the sealed container are fully stirred and heated to 105-200 DEG C under high temperature and high pressure.

5. The method of claim 4, wherein the extraction of GLP-4 is performed by using water as the extraction solvent. In step S2, the Ganoderma lucidum powder and water in the sealed container are fully stirred and heated to 105-200 DEG C under high temperature and high pressure.

6. The method of claim 5, wherein the extraction of Ganoderma lucidum polysaccharide GLP-4 is characterized by, In step S6, the freeze-dried powder containing medicinal ingredients is prepared into 40-80 mg / ml by adding pure water, and separated by column chromatography multiple times.

7. The method of claim 6, wherein the extraction of Ganoderma lucidum polysaccharide GLP-4 is characterized by, In step S4, the medicinal residues are mixed with a NaOH solution according to a predetermined ratio, soaked at a temperature of 40-100 DEG C, and then neutralized by adding HCl, wherein the soaking time is 1-5 h, and the concentration of NaOH is 0.05-0.5 mol / L. 8.The method of claim 7, wherein the extraction of the Ganoderma lucidum polysaccharide GLP-4 is characterized by, In step S1, the Ganoderma lucidum is washed with clean water to remove surface dust, dried at 105 DEG C, and then crushed, and the crushed Ganoderma lucidum powder is greater than 60 mesh.

9. The method of claim 8, wherein the extraction of Ganoderma lucidum polysaccharide GLP-4 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, 140 DEG C, 145 DEG C, 150 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, and boiled for 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h or 6 h, and the internal pressure of the sealed container gradually increases with the increase of the heating temperature to form a high temperature and high pressure environment in the sealed container.

10. The use of Ganoderma lucidum polysaccharide GLP-4 according to claim 1 or 2, characterized in that, The Ganoderma lucidum polysaccharide GLP-4 has good water-soluble performance, is easily absorbed by the human body, has the effects of treating emphysema, pulmonary fibrosis and anti-tumor, especially the treatment effects on emphysema and pulmonary fibrosis are exceptionally excellent, and is obviously superior to bifenox, and when it is used in combination with a chemotherapy drug, it can eliminate the toxic and side effects of the chemotherapy drug on the human body and has a certain effect on controlling, reducing and eliminating tumor cells.

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