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

Abstract

The present invention is suitable for the technical field of plant extraction and separation, and provides a polysaccharide compound having a definite molecular structure. The polysaccharide compound is suitable for treatment of patients suffering from advanced cancers (no longer satisfying the operation condition, only three to six months of survival time being left, but still satisfying the chemotherapeutic condition). The present invention comprises: S1, carrying out dust removal on Ganoderma lucidum, drying same and crushing same; S2, putting same into a closed container, mixing same with water, and performing high-temperature and high-pressure extraction to obtain a pharmaceutical effect ingredient; S3, removing dregs form an extracted concentrated liquid containing the pharmaceutical effect ingredient; and S4, preparing the dreg-removed concentrated liquid into a solution having a certain concentration, and performing column chromatography separation to obtain the pharmaceutical effect ingredient a polysaccharide. The pharmaceutical effect ingredient polysaccharide has good water solubility and is easy to absorb by the human body, has an anti-tumor pharmaceutical effect, and has an effect of preventing tumorigenesis for humans. Especially, during use in combination with chemotherapeutic drugs, the polysaccharide can eliminate the toxic and side effects of the chemotherapeutic drugs on the human body, and control and reduce tumor lumps and reduce and kill tumor cells, which are all proved by corresponding experimental data.

Description

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

[0001] The present invention belongs to the field of extraction and separation technology improvement, and in particular relates to a method for extracting Ganoderma lucidum polysaccharide GLP-1 and its application. Background Art

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

[0003] Summary of the Invention

[0004] The purpose of the present invention is to provide an improved method for extracting polysaccharide compounds with clear molecular structure and pharmacological function from Ganoderma lucidum raw materials and its application.

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

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

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

[0008] The present invention is achieved by providing a method for extracting GLP-1 polysaccharide from Ganoderma lucidum, the method comprising the following steps:

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

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

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

[0012] S4. The concentrated solution containing the medicinal component is mixed with pure water in a ratio of a preset concentration to obtain an aqueous solution through multiple column chromatography separation techniques to obtain Ganoderma lucidum polysaccharide GLP-1 containing the medicinal component.

[0013] A further technical solution of the present invention is: in step S2, the mixed liquid of Ganoderma lucidum powder and water in the sealed container is fully stirred and then heated to 105-200° C., and the boiling time is continued for 2-6 hours. As the heating temperature increases, the pressure inside the sealed container gradually increases to form a high-temperature and high-pressure environment in the sealed container.

[0014] A further technical solution of the present invention is: in step S2, the mixed liquid of Ganoderma lucidum powder and water in the sealed container is heated to 105-170° C. and the boiling time is continued for 3-6 hours. As the heating temperature increases, the pressure inside the sealed container gradually increases to form a high-temperature and high-pressure environment in the sealed container.

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

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

[0017] A further technical solution of the present invention is: in step S1, the Ganoderma lucidum is washed with clean water to remove dust on the surface and then dried at 105° C. The dried Ganoderma lucidum is crushed, and the crushed Ganoderma lucidum powder is larger than 60 mesh.

[0018] A further technical solution of the present invention is: in the step S2, the mixed liquid in the closed container is heated to 105°C, 110°C, 115°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, 170°C, 175°C, 180°C, 185°C, 190°C, 195°C or 200°C, and the boiling time is 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h, and the pressure inside the closed container gradually increases as the heating temperature increases, forming a high temperature and high pressure environment of the closed container.

[0019] Another object of the present invention is to provide a Ganoderma lucidum polysaccharide GLP-1, the structural formula of the Ganoderma lucidum polysaccharide GLP-1 is

[0020] Molecular formula (C 60 H 100 O 50 )n, where n=17-25.

[0021] A further technical solution of the present invention is: n is 17, 18, 19, 20, 21, 22, 23, 24 or 25.

[0022] Another object of the present invention is to provide an application of Ganoderma lucidum polysaccharide GLP-1, which has good water solubility, is easily absorbed by the human body, has anti-tumor efficacy and is effective in preventing the occurrence of tumors in humans. Especially when used in combination with chemotherapy drugs, it can eliminate the toxic side effects of chemotherapy drugs on the human body and is effective in controlling and reducing tumor masses and reducing and eliminating tumor cells.

[0023] The beneficial effects of the present invention are: the method has a simple extraction process, a high polysaccharide extraction rate, a low production cost, and is easy to industrialize; the Ganoderma lucidum polysaccharide GLP-1 has good solubility and is easily absorbed by the human body, thereby exerting its anti-tumor, antioxidant, and anti-aging effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG1 is a flow chart of a method for extracting GLP-1, a polysaccharide of Ganoderma lucidum, provided in an embodiment of the present invention.

[0025] FIG2 is a schematic diagram of a 1gMp-RT (peak molecular weight) calibration curve provided in an embodiment of the present invention.

[0026] FIG3 is a schematic diagram of a 1gMp-RT (weight average molecular weight) calibration curve provided in an embodiment of the present invention.

[0027] FIG4 is a schematic diagram of a 1gMp-RT (number average molecular weight) calibration curve provided in an embodiment of the present invention.

[0028] FIG5 is a schematic diagram of a molecular weight spectrum of a sample provided in an embodiment of the present invention.

[0029] FIG6 is a schematic diagram of an ion chromatogram of a mixed standard of 16 sugars provided in an embodiment of the present invention.

[0030] FIG7 is a second schematic diagram of an ion chromatogram of a mixed standard of 16 sugars provided in an embodiment of the present invention.

[0031] FIG8 is a GCMS chromatogram of a sample (PMAA) provided in an embodiment of the present invention.

[0032] FIG9 is a schematic diagram 1 of the result analysis of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0033] FIG10 is a second schematic diagram of the analysis results of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0034] FIG11 is a third schematic diagram of the analysis results of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0035] FIG12 is a fourth schematic diagram of the analysis results of polysaccharide methylated alditol acetyl ester (PMAA) provided in an embodiment of the present invention.

[0036] FIG13 is a schematic diagram of a hydrogen spectrum provided by an embodiment of the present invention.

[0037] FIG14 is a schematic diagram of a carbon spectrum provided by an embodiment of the present invention.

[0038] FIG15 is a schematic diagram of a Dept135 spectrum provided in an embodiment of the present invention.

[0039] FIG16 is a schematic diagram of HH-COSY provided by an embodiment of the present invention.

[0040] FIG17 is a HSQC diagram provided by an embodiment of the present invention.

[0041] FIG18 is a HMBC spectrum provided by an embodiment of the present invention.

[0042] FIG19 is a schematic diagram of NOESY provided by an embodiment of the present invention.

[0043] FIG20 is a comparative schematic diagram of the effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the tumor volume of Lewis cell tumor-bearing mice provided by an embodiment of the present invention.

[0044] 21 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on tumor pathology in Lewis cell tumor-bearing mice (×200) provided by an embodiment of the present invention.

[0045] Figure 22 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on renal pathology in Lewis cell tumor-bearing mice (×200) provided by an embodiment of the present invention.

[0046] 23 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on gastric pathology in Lewis cell tumor-bearing mice (×200) provided by an embodiment of the present invention.

[0047] 24 is a schematic diagram showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on spleen pathology in Lewis cell tumor-bearing mice (×200) provided in an embodiment of the present invention.

[0048] 25 is a schematic diagram showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on the tumor volume of Lewis cell tumor-bearing mice provided by an embodiment of the present invention.

[0049] 26 is a schematic diagram showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on the thymus tissue pathology of Lewis cell tumor-bearing mice (×200) provided by an embodiment of the present invention.

[0050] 27 is a schematic diagram showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on spleen tissue pathology in Lewis cell tumor-bearing mice (×200) provided by an embodiment of the present invention.

[0051] FIG28 is a schematic diagram showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on nasal tissue pathology in Lewis cell tumor-bearing mice (×200) provided by an embodiment of the present invention.

[0052] 29 is a schematic diagram showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on gastric tissue pathology in Lewis cell tumor-bearing mice (×200) provided by an embodiment of the present invention.

[0053] FIG30 is a schematic diagram showing the effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on the renal tissue pathology of Lewis cell tumor-bearing mice (×200) provided by an embodiment of the present invention.

[0054] Figures 31, 32, 33, and 34 are schematic diagrams of mice showing the effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy provided by an embodiment of the present invention on H22 liver cancer-bearing mice.

[0055] Figures 35, 36, 37 and 38 are schematic diagrams of the appearance of tumors in H22 liver cancer-bearing mice, showing the effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy provided by an embodiment of the present invention.

[0056] Figures 39, 40, 41 and 42 are schematic diagrams of mice showing the effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on 4T1 breast cancer tumor-bearing mice provided by the embodiments of the present invention.

[0057] Figures 43, 44, 45 and 46 are schematic diagrams of the tumor appearance of the effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on 4T1 breast cancer-bearing mice provided by the embodiments of the present invention.

[0058] Figures 47, 48, 49, 50, 51 and 52 are schematic diagrams showing the effects of Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin on LLC lung cancer cells provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0059] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0060] As shown in FIG1 , the flow chart of the method for extracting GLP-1, a polysaccharide of Ganoderma lucidum provided by the present invention, is described in detail as follows:

[0061] Step S1, cleaning the harvested Ganoderma lucidum or Ganoderma lucidum that has undergone a preliminary simple treatment with clean water on a cleaning device, removing dust from the surface thereof, transferring the Ganoderma lucidum to a drying device for drying at a drying temperature of 105° C., placing the dried Ganoderma lucidum on a crusher for crushing into Ganoderma lucidum powder, sieving the Ganoderma lucidum powder, transferring the Ganoderma lucidum powder particles larger than 60 mesh to the next process, and returning the Ganoderma lucidum powder particles smaller than 60 mesh to the crushing mechanism for re-crushing, repeating the process multiple times until the crushed Ganoderma lucidum powder particles meet the specified requirements.

[0062] Step S2: mixing the qualified Ganoderma lucidum powder with pure water and placing the mixture in a sealed container, heating the sealed container at a high temperature. During the heating process, the pressure inside the sealed container gradually increases as the temperature rises, so that a high temperature and high pressure environment is formed in the sealed container. In this environment, it is easier to fully fuse the Ganoderma lucidum powder and water to form a mixed liquid. The sealed container is heated to a temperature between 105° C. and 200° C. for a boiling time of 2 to 6 hours, preferably between 105° C. and 170° C. The boiling time is 3-6 hours, and it is more preferably heated to 105°C, 110°C, 115°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, 170°C, 175°C, 180°C, 185°C, 190°C, 195°C or 200°C, and the boiling time is 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h, so that the mixed liquid is fully fused.

[0063] In step S3, the extracted aqueous solution containing the medicinal components is centrifuged to remove the residue, and the centrifuged liquid is concentrated by a membrane to obtain a concentrated liquid.

[0064] Step S4, preparing the concentrated solution containing the medicinal ingredients to a certain concentration and separating it by column chromatography to obtain Ganoderma lucidum polysaccharide GLP-1 containing the medicinal ingredients.

[0065] The method has simple extraction process, high polysaccharide extraction rate, low production cost and simple operation.

[0066] Another object of the present invention is to provide a Ganoderma lucidum polysaccharide GLP-1, the structural formula of the Ganoderma lucidum polysaccharide GLP-1 is

[0067] Molecular formula (C 60 H 100 O 50 )n, where n=17-25.

[0068] n is 17, 18, 19, 20, 21, 22, 23, 24 or 25.

[0069] After obtaining the Ganoderma lucidum polysaccharide GLP-1 with the above structure, it was subjected to determination experiments and the following reported results were obtained.

[0070] 1. Molecular weight determination

[0071] 1. Purpose of the experiment

[0072] The molecular weight and purity of the polysaccharide were determined by HPGPC.

[0073] 2. Experimental Materials

[0074] 2.1 Instruments

[0075] 2.2 Materials

[0076] 2.3 Standards

[0077] 3. Experimental Procedure

[0078] 3.1 Reagent preparation

[0079] 3.2 Preparation of sample and standard solutions

[0080] Accurately weigh the sample and standard, prepare the sample into a 5 mg / ml solution, centrifuge at 12000 rpm for 10 min, filter the supernatant with a 0.22 μm microporous filter membrane, and then transfer the sample to a 1.8 ml injection vial.

[0081] 3.3 Chromatographic method

[0082] Chromatographic column: BRT105-104-102 gel column in series (8×300 mm); mobile phase: 0.05 M NaCl solution; flow rate: 0.6 ml / min, column temperature: 40°C; injection volume: 20 μl; detector: differential detector RI-10A.

[0083] 4. Experimental Results

[0084] As shown in Figure 2-4, the calibration curves of 1gMp-RT (peak molecular weight), 1gMw-RT (weight average molecular weight), and 1gMn-RT (number average molecular weight) were obtained.

[0085] The equation of the 1gMp-RT calibration curve is: y = -0.1802x + 11.661R2 = 0.9928;

[0086] The equation of the lgMw-RT calibration curve is: y = -0.1926x + 12.241R2 = 0.9965;

[0087] The equation of the lgMn-RT calibration curve is: y = -0.1783x + 11.506R2 = 0.9911;

[0088] Based on the standard curve, a calculation formula was derived to calculate the molecular weight of each sample. The molecular weight spectrum of the samples is shown in Figure 5, and the calculation results are shown in the table below.

[0089] Among them, 46.5min is the peak of the mobile phase.

[0090] 2. Monosaccharide composition determination experiment

[0091] 1. Purpose of the experiment

[0092] The monosaccharide composition was determined using ion chromatography.

[0093] 2. Experimental Principle

[0094] Based on the electrochemical activity of carbohydrate molecules and their ionization in strong alkaline solutions, carbohydrate compounds are weak acids with a pKa greater than 11. In high-pH eluents, they exist partially or completely as anions. By taking advantage of the differences in ion exchange caused by the pKa of different carbohydrate compounds and the different hydrophobic interactions between certain carbohydrates and anion exchange resins, efficient anion exchange separation of carbohydrate compounds is achieved. The current generated by the oxidation reaction of hydroxyl groups in the carbohydrate molecular structure on the gold electrode surface is then detected.

[0095] 3. Experimental Materials

[0096] 3.1 Instruments

[0097] 3.2 Reagents

[0098] 3.3 Standards

[0099] 4. Experimental Methods

[0100] 4.1 Reagent preparation

[0101] 4.2 Preparation and calculation method of standard solution

[0102] Sixteen monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid, mannuronic acid) were prepared into standard stock solutions.

[0103] Prepare a standard concentration of each monosaccharide standard solution as a mixed standard. Determine the mass of each monosaccharide using the absolute quantification method, and calculate the molar ratio based on the molar mass of the monosaccharides.

[0104] 4.3 Sample preparation

[0105] Accurately weigh 5 mg of sample into an ampoule, add 2 ml of 3M TFA, and hydrolyze at 120°C for 3 h. Accurately transfer the acid hydrolysis solution to a tube and blow dry with nitrogen. Add 5 ml of water and vortex to mix thoroughly. Aspirate 50 μL and add 950 μL of deionized water. Centrifuge at 12,000 rpm for 5 min. Collect the supernatant for IC analysis.

[0106] 4.4 Chromatographic methods

[0107] Chromatographic column: Dionex CarbopacTM PA20 (3*150mm); mobile phase: A: H2O; B: 15mMNaOHC: 15mMNaOH&100mM NaOAC; flow rate: 0.3ml / min; injection volume: 5μL; column temperature: 30℃; detector: electrochemical detector.

[0108] 4.5 Standard Sequence

[0109] C(standard) / A(standard)=C(sample) / A(sample)

[0110] 5. Experimental Results

[0111] Mixed standard: Solvent peak: 2.0 min is the peak of sodium hydroxide, 41 min is the peak of sodium acetate, as shown in Figure 6.

[0112] 3. Polysaccharide Linkage Determination Experiment

[0113] 1. Purpose of the experiment

[0114] The polysaccharide samples were derivatized by methylation and then their connection patterns were determined by GC-MS.

[0115] 2. Experimental Materials

[0116] 2.1 Instruments

[0117] 2.2 Reagents

[0118] 3. Experimental Methods

[0119] 3.1 Reagent preparation

[0120] 3.2 Sample methylation

[0121] After methylation, hydrolysis and acetylation, the samples were determined by GC-MS and compared with the standard mass spectrum library.

[0122] Weigh a polysaccharide sample (2-3 mg) into a glass reaction vial. Add 1 mL of anhydrous DMSO and quickly add methylation reagent solution A. Seal the vial, dissolve under ultrasound, and then add methylation reagent solution B. React in a magnetically stirred water bath at 30°C for 60 minutes. Finally, add 2 mL of ultrapure water to terminate the methylation reaction.

[0123] Take the methylated polysaccharide and add 1 ml of 2 M trifluoroacetic acid (TFA) for hydrolysis for 90 minutes, then evaporate to dryness on a rotary evaporator. Add 2 ml of double-distilled water and 60 mg of sodium borohydride to the residue for reduction for 8 hours, neutralize with glacial acetic acid, rotary evaporate, and oven dry at 101°C. Then, add 1 ml of acetic anhydride for acetylation at 100°C for 1 hour, then cool. Then, add 3 ml of toluene, concentrate under reduced pressure, and evaporate to dryness. Repeat this process 4-5 times to remove excess acetic anhydride.

[0124] The acetylated product was dissolved in 3 mL of CH₂Cl₂ and transferred to a separatory funnel. A small amount of distilled water was added and shaken thoroughly, followed by removal of the upper aqueous layer. This process was repeated four times. The CH₂Cl₂ layer was dried over an appropriate amount of anhydrous sodium sulfate, brought to a volume of 10 mL, and placed in a liquid phase vial. The acetylated product samples were analyzed using a Shimadzu GCMS-QP 2010 gas chromatograph-mass spectrometer.

[0125] GC-MS conditions: RXI-5SIL MS column 30m*0.25mm*0.25um; temperature program conditions: starting temperature 120℃, heating to 250℃ / min at 3℃ / min; hold for 5min; injection port temperature 250℃, detector temperature 250℃, carrier gas helium, flow rate 1mL / min.

[0126] 4. Experimental Results

[0127] The GCMS chromatogram of the sample (PMAA) is shown in Figure 8.

[0128] The results of polysaccharide methylated alditol acetyl ester (PMAA) are shown in the table below and in Figures 11-14.

[0129] 4. NMR analysis spectrum and analysis

[0130] 1.1. Experimental Materials and Instruments

[0131] Heavy water (D2O, 99.9%) and deuterated acetone were used as internal references; freeze dryer and nuclear magnetic resonance (NMR) instrument were Bruker 600M.

[0132] 2. Experimental Procedure

[0133] Weigh 50 mg of the polysaccharide sample, dissolve it in 0.5 ml of deuterated water, and freeze-dry it. The freeze-dried powder is then dissolved in 0.5 ml of deuterated water and freeze-dried again, repeating the process to fully exchange the active hydrogen. The sample is then dissolved in 0.5 ml of deuterated water and subjected to measurement at room temperature (25°C) on a 600 MHz nuclear magnetic resonance instrument for 1H NMR, 13C NMR, and DEPT135 one-dimensional and two-dimensional spectra.

[0134] 3. Experimental Results

[0135] The hydrogen spectrum signals are mainly concentrated between 3.0 and 5.5 ppm. δ3.2-4.0 ppm is the sugar ring proton signal, and the main terminal proton peaks at δ4.43, 4.45, 4.47, and 4.66 are concentrated in the 4.3-5.5 ppm region, as shown in Figure 13.

[0136] Carbon spectrum analysis 13 C NMR (201 MHz, D2O): The NMR carbon spectrum signals are primarily concentrated between 60-120 ppm. By observing the carbon spectrum, we can see that the main anomeric carbon signal peaks at δ103.81, 103.82, 103.88, and 104.13 are primarily located between δ93 and 105. Furthermore, the δ76.68, 74.08, 82.67, 76.62, 70.19, 74.15, 71.56, 80.14, 76.67, 61.66, 74.75, 85.63, 77.17, 76.97, 62.03, 74.76, 74.35, 77.31, 69.61, and 62.04 ppm regions are also present. Based on the monosaccharide composition, the polysaccharide is composed of glucose, indicating that it is primarily dextran. This is shown in Figure 14.

[0137] Dept 135 spectrum analysis showed that the peaks at 70.19, 61.66, 62.03, and 62.04 ppm were inverted peaks, indicating chemical shifts of C6, as shown in Figure 15 .

[0138] As shown in Figures 16-19, the anomeric carbon signal is observed at δ104.13 through HSQC, and the corresponding anomeric hydrogen signal is δ4.66 in the HSQC spectrum. Using HH-COSY, the signals for H1-2 are 4.66 / 3.3; H2-3 are 3.3 / 3.42; and H3-4 are 3.42 / 3.66. We can infer that H1, H2, H3, and H4 have δ4.66, 3.3, 3.42, and 3.66, respectively. The corresponding δs are 104.13, 74.76, 74.35, and 77.31. Using HH-COSY, the signals for H6b-6a are 3.81 / 3.62; and H6a-5 are 3.62 / 3.46. We can infer that H6b, H6a, and H5 have δ3.81, 3.62, and 3.46, respectively. The corresponding C6 and C5 are δ62.04 and 74.54, respectively. Therefore, this signal should be attributed to the glycosidic bond β-Glcp-(1→

[0139] Through the HSQC spectrum, we can observe that the anomeric carbon signal is δ103.81, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ4.45. Through HH-COSY, the signals of H1-2 are 4.45 / 3.25; the signals of H2-3 are 3.25 / 3.42; the signals of H3-4 are 3.42 / 3.64; we can infer that the signals of H1, H2, H3, and H4 are δ4.45, 3.25, 3.42, 3.64, and its corresponding C1-4 are δ103.81, 76.68, 74.08, and 82.67; and Dept135 combined with HSQC can determine that δ3.78 and 4.13 are attributed to the peaks of H6a and b, and the corresponding C6 is 70.19ppm. In the HMBC diagram, H6b intersects with C5 and C4 at 3.78 / 76.62 and 3.78 / 82.67, respectively, so C5 is 76.62ppm. The corresponding H5 by HSQC is 3.82ppm. Therefore, this signal should be attributed to the glycosidic bond →4,6)-β-D-Glcp-(1→

[0140] The anomeric carbon signal was observed at δ103.88 by HSQC, and the corresponding anomeric hydrogen signal was δ4.47 by HSQC. By HH-COSY, the signals of H1-2 were 4.47 / 3.47, and those of H2-3 were 3.47 / 3.65. We can infer that H1, H2, and H3 were δ4.47, 3.47, and 3.65, respectively, and their corresponding C1-3 values ​​were δ103.88, 74.75, and 85.63. In the HMBC spectrum, 85.63 / 3.44 and 76.97 / 3.71 were assigned to C3 / H5 and C5 / H4. Dept135 combined with HSQC determined that δ3.65 and 3.83 were assigned to the peaks of H6a,b, and the corresponding C6 value was 62.03 ppm. Therefore, this signal should be attributed to the glycosidic bond →3,4)-β-D-Glcp-(1→

[0141] The anomeric carbon signal was observed at δ103.82 by HSQC, and the corresponding anomeric hydrogen signal was δ4.43 by HSQC. The signals of H1-2 were 4.43 / 3.28 by HH-COSY, and those of H2-3 were 3.28 / 3.44. We can infer that H1, H2, and H3 were δ4.43, 328, and 3.44, respectively, and their corresponding C1-3 values ​​were δ103.82, 74.15, and 76.82. C4 shifted downfield, and 80.14 ppm was assigned to C4. Dept135 combined with HSQC determined that δ3.68 and 3.84 were assigned to the peaks of H6a,b, and the corresponding C6 value was 61.66 ppm. Therefore, this signal should be attributed to the glycosidic bond →4)-β-D-Glcp-(1→

[0142] According to similar rules and combined with HMBC and NOESY, all glycosidic bond signals are assigned, as shown in the following table: Hydrogen and carbon signal assignment

[0143] Main chain analysis:

[0144] In the HMBC spectrum, we attributed the glycosidic bond signals of the polysaccharide based on the one-dimensional and two-dimensional NMR spectra; the anomeric carbon of the glycosidic bond →4)-β-D-Glcp-(1→ and its own H4 have related signal peaks, indicating the existence of a linking mode of →4)-β-D-Glcp-(1→4)-β-D-Glcp-(1→.

[0145] The anomeric hydrogen of the glycosidic bond →4)-β-D-Glcp-(1→ and the C4 of →3,4)-β-D-Glcp-(1→) have related signal peaks, indicating the existence of the linking mode of →4)-β-D-Glcp-(1→3,4)-β-D-Glcp-(1→.

[0146] The anomeric hydrogen of the glycosidic bond →3,4)-β-D-Glcp-(1→ and the C4 of its →4,6)-β-D-Glcp-(1→ have related signal peaks, indicating the existence of the →3,4)-β-D-Glcp-(1→4,6)-β-D-Glcp-(1→ linking mode.

[0147] Branch chain analysis:

[0148] There are related signal peaks between the anomeric hydrogen of the glycosidic bond β-D-Glcp-(1→) and C3 of its →3,4)-β-D-Glcp-(1→, indicating the existence of the β-D-Glcp-(1→3,4)-β-D-Glcp-(1→ linking mode.

[0149] There are related signal peaks between the anomeric carbon of the glycosidic bond β-D-Glcp-(1→4,6)-β-D-Glcp-(1→ H6, indicating the existence of the β-D-Glcp-(1→4,6)-β-D-Glcp-(1→ linking mode.

[0150] In summary, we can infer that the main chain of this polysaccharide is β-1,4 glucan, and β-D-Glcp-(1→) is connected to the main chain through the o-3 of →3,4)-β-D-Glcp-(1→) and the o-6 of →4,6)-β-DGlcp-(1→. Its simplified structure is shown below.

[0151] Another object of the present invention is to provide an application of Ganoderma lucidum polysaccharide GLP-1, which has good water solubility, is easily absorbed by the human body, has anti-tumor efficacy and has a strong effect on preventing the occurrence of tumors in humans. Especially when used in combination with chemotherapy drugs, it can eliminate the toxic side effects of chemotherapy drugs on the human body and has the effect of controlling, reducing tumor masses and reducing and eliminating tumor cells.

[0152] The Ganoderma lucidum polysaccharide GLP-1 is used in drugs for inhibiting tumor spread or enhancing human immunity.

[0153] Experimental study and data on the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on Lewis lung cancer-bearing mice

[0154] Purpose of the experiment

[0155] Lewis lung cancer cells were inoculated in the right axilla of C57 mice to prepare tumor-bearing mice. The anti-tumor effect of Ganoderma lucidum polysaccharide GLP-1 on lung cancer-bearing mice was studied to provide experimental basis for its clinical research.

[0156] Experimental Materials

[0157] Test sample

[0158] Ganoderma lucidum polysaccharide GLP-1, batch number: ALM20200518A1; provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0159] Positive control

[0160] Kanglaite soft capsules, batch number: 20200802, product of Zhejiang Kanglaite Pharmaceutical Co., Ltd.; cisplatin injection, batch number: 601210104, product of Jiangsu Hausen Pharmaceutical Group Co., Ltd.

[0161] experimental animals

[0162] 80 SPF male C57 mice, weighing 12-15 g, were provided by Hunan Slake Jingda Experimental Animal Co., Ltd., with experimental animal production license number: SCXK (Xiang) 2019-0004 and experimental animal quality certificate number: 430727211103153225. They were raised in Area D of the barrier environment animal laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd., with experimental animal use license number: SYXK (Xiang) 2020-0015.

[0163] Main reagents

[0164] 0.9% sodium chloride injection, batch number: 21071401C, product of Hunan Kangyuan Pharmaceutical Co., Ltd.; ALP assay kit, batch number: 201223, purchased from Mike Biotech Co., Ltd.; TP assay kit, batch number: 105467, ALB assay kit, batch number: 105468, ALT assay kit, batch number: 105475, AST assay kit, batch number: 104446, TBIL assay kit, batch number: 105465, GGT assay kit, batch number: 104447, all are products of Wako Pure Chemical Industries, Ltd.

[0165] Main instruments

[0166] S10 portable high-speed disperser, Shanghai Xinzhi; AR223CN electronic balance, Ohaus Instruments (Changzhou) Co., Ltd.; LABOSPECT003 automatic biochemical analyzer, Hitachi, Japan; TDZ5-WS desktop multi-tube automatic balancing centrifuge, Hunan Kaida Industrial Development Co., Ltd.; ME2002E electronic balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.; digital display vernier caliper, Yongkang Zhengfeng Hardware Co., Ltd.; flow cytometer, BD; ASP200S fully automatic tissue dehydrator, ASP300S fully automatic tissue dehydrator, TP1020 fully automatic dehydrator, HI1210 slide spreader, HI1220 slide baking machine, RM2235 paraffin slicer, EG1150H+C tissue embedding machine, AutoStainer XL automatic slide stainer + CV5030 automatic coverslip machine, Leica, Germany; BX43 biological microscope + MD50 digital imaging system, CX31 biological microscope, Olympus, Japan.

[0167] Experimental methods

[0168] 1×10 Lewis tumor cells in logarithmic growth phase were taken 7 0.2 mL of the solution was inoculated into the right axilla of 8 normal male C57 mice. The tumor growth was observed and the mice were prepared into tumor-bearing mice. When the tumor volume of the tumor-bearing mice reached 200 mm 3 At the above time, the tumor tissue of the tumor-bearing mice was aseptically removed to prepare a tissue cell suspension, and the tumor tissue homogenate was made with cell culture medium at a ratio of 1:1 (V:V). The suspension was inoculated into the right axilla of 62 male C57 mice with an inoculation volume of 0.2 mL per mouse. The suspension was kept for 24 h until the tumor volume of the tumor-bearing mice reached 100 mm. 3 At the above time, mice bearing tumors without ulceration and with good growth were selected and randomly divided into groups according to tumor volume: model control group, cisplatin group (4 mg / kg), Kanglaite soft capsule group (1404 mg / kg), cisplatin + Kanglaite soft capsule group (4 + 1404 mg / kg), cisplatin + GLP-1 low-dose group (4 + 130 mg / kg), cisplatin + GLP-1 high-dose group (4 + 1170 mg / kg), 8 mice in each group, and 8 mice as normal control group. Cisplatin in the cisplatin group and the combination group was administered once every 3 days, Kanglaite was administered once a day, and GLP-1 was administered once a day. The dosage volume was 20 mL / kg (oral gavage) and 10 mL / kg (intraperitoneal injection), respectively, and the administration was continued for 27 days. After the last dose, blood was collected from the orbital cavity to test WBC, RBC, liver and kidney function (ALT, AST, BUN, CRE), and CD4+ and CD8+ levels. The spleen, thymus, and tumor were weighed and the organ-to-body ratio was calculated. Bone marrow was collected for smear examination, and tumors, spleen, stomach, and kidneys were subjected to histopathological analysis.

[0169] Dosage design

[0170] Ganoderma lucidum polysaccharide GLP-1 was designed based on the results of previous studies. The low dose was 130 mg / kg and the high dose was 1170 mg / kg. The corresponding drugs were administered as shown in Table 1. This experiment was conducted at the same multiple of the intended clinical dose.

[0171] The proposed clinical dosage of Kanglaite Soft Capsules is 0.45g / capsule, 6 capsules / time, 4 times / day, which is 10.8g / day. The equivalent dose in mice based on body surface area is 10.8g / day*0.0026 / 0.02kg=1404mg / kg. This trial was conducted at the same multiple of the proposed clinical dosage.

[0172] Cisplatin dosage design basis: According to the clinical dosage of cisplatin, each person should not exceed 100 mg / m2 per day. 2 The tolerance of mice to cisplatin was evaluated, and a dose of 4 mg / kg was selected as the administration dose.

[0173] Table 1 Trial grouping and dosage design

[0174] Detection indicators

[0175] Efficacy indicators

[0176] Evaluation of animal quality of life and survival time: During the experiment, the weight of each group of animals, animal mortality, mental state, coat color, coat smoothness, stool form, and the presence of diarrhea were recorded in detail.

[0177] Tumor volume detection: After administration, the length and width of the mouse tumor were measured with a vernier caliper every 3 days to calculate the tumor volume. Tumor volume (TV) = 1 / 2 × a (length) × b 2 (width). Relative tumor volume (RTV) = TV t / TV0, TV0 is the tumor volume at the time of drug administration, TV t is the tumor volume measured each time. Relative tumor proliferation rate T / C (%) = T RTV / C RTV ×100, T RTV :RTV in treatment group; C RTV : RTV of model control group. Tumor growth inhibition rate (%) = (tumor weight of model control group - tumor weight of drug-treated group) / tumor weight of model control group × 100%. A tumor growth inhibition rate (%) < 40% was considered ineffective; a tumor growth inhibition rate (%) ≥ 40% and P ≤ 0.05 was considered effective.

[0178] Spleen and thymus organ coefficients: After the last administration, the spleen, thymus, and tumor were weighed and the organ coefficients were calculated. Organ coefficient (%) = organ weight / fasting body weight × 100%.

[0179] Reduce toxicity and increase efficacy

[0180] Hematological tests: After the last dose, blood was collected from the orbital cavity for routine blood tests (WBC, RBC) and liver and kidney function indicators (BUN, CRE).

[0181] Bone marrow examination: After the last administration, bone marrow was taken for smear and the changes of bone marrow cells in each group of mice were observed.

[0182] Histopathological examination: After the last administration, tumor tissues, spleen, stomach, and kidney were collected for HE staining to observe the histopathological changes.

[0183] Blood CD4+ and CD8+ content detection: After the last administration, blood lymphocyte typing CD4+ and CD8+ content were detected by flow cytometry.

[0184] Data processing and statistical analysis

[0185] Data were rounded to the nearest integer and statistically analyzed according to our center's standard operating procedures. SPSS software was used for statistical analysis. Measurement data were presented as mean ± standard deviation (xx ± s). Leven's test was used to test for normality and homogeneity of variance. If the difference was not statistically significant (P>0.05), one-way analysis of variance (ANOVA) was used for statistical analysis. If the ANOVA was statistically significant (P≤0.05), the LSD test (parametric method) was used for comparative analysis. If the variance was not homogeneous (P≤0.05), the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P≤0.05), the Dunnett's test (nonparametric method) was used for comparative analysis. Statistical results were tested with an α = 0.05 threshold, with P≤0.05 indicating statistical significance and P≤0.01 indicating highly significant differences.

[0186] Experimental results

[0187] General clinical observations and animal deaths

[0188] Before administration, mice were active, with normal movements and gait. Model control group mice 2M06 and 2M03 died on January 20, 2022, and January 21, 2022, respectively; Kanglaite soft capsule group mice 4M01 died on January 19, 2022; cisplatin + Kanglaite soft capsule group mice 5M01 and 5M04 died on January 14, 2022, and January 22, 2022, respectively; and cisplatin + high-dose GLP-1 group mice 7M08 died on January 9, 2022.

[0189] The mortality rates of each group were 0%, 25.0%, 0%, 12.5%, 25.0%, 0%, and 12.5%, respectively.

[0190] Table 2 Statistics of the number of surviving animals and mortality rates in each group

[0191] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on body weight in Lewis lung cancer-bearing mice

[0192] As shown in Table 3, compared with the normal control group, the body weight of mice in the model control group was significantly reduced on Day 4 after administration (P ≤ 0.05). Compared with the model control group, the body weight of mice in the cisplatin group and the cisplatin combined with Kanglaite soft capsule group was significantly reduced on Day 7 to Day 25 after administration (P ≤ 0.01); the body weight of mice in the cisplatin combined with low- and high-dose GLP-1 groups was significantly reduced on Day 4 to Day 25 after administration (P ≤ 0.05 or P ≤ 0.01); and the body weight of mice in the Kanglaite soft capsule group was significantly reduced on Day 10 to Day 19 after administration.

[0193] Compared with the cisplatin group, the body weight of mice in the Kanglaite soft capsule group increased significantly from Day 7 to Day 25 (P ≤ 0.05 or P ≤ 0.01), while the body weight of mice in the cisplatin combined with GLP-1 high-dose group decreased significantly from Day 7 to Day 25 (P ≤ 0.05). Compared with the Kanglaite soft capsule group, the body weight of mice in the cisplatin combined with Kanglaite soft capsule group and the low- and high-dose GLP-1 groups decreased significantly from Day 7 to Day 25 (P ≤ 0.05 or P ≤ 0.01). Compared with the cisplatin + Kanglaite soft capsule group, there was no statistically significant difference between the groups.

[0194] [Corrected 27.03.2024 according to Rule 91] Table 3 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the body weight of Lewis cell tumor-bearing mice 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 Kanglaite soft capsule group, & P≤0.05, && P≤0.01, compared with cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0195] [Corrected 27.03.2024 in accordance with Article 91]

[0196] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on tumor volume and relative tumor volume in Lewis lung cancer-bearing mice

[0197] As shown in Table 4, compared with the model control group, the tumor volume of mice in the low- and high-dose cisplatin combined with GLP-1 groups was significantly reduced from D10 to D25 (P ≤ 0.05 or P ≤ 0.01). The tumor volume of mice in the cisplatin group was significantly reduced from D10 and D16 to D25 (P ≤ 0.05 or P ≤ 0.01). The tumor volume of mice in the cisplatin combined with Kanglaite soft capsule group was significantly reduced from D13 to D25 (P ≤ 0.05 or P ≤ 0.01). There was no significant difference in tumor volume in the Kanglaite soft capsule group. Compared with the cisplatin group, the tumor volume of mice in the high-dose cisplatin combined with GLP-1 group was significantly reduced from D22 to D25 (P ≤ 0.05), while the tumor volume of mice in the Kanglaite soft capsule group was significantly increased from D7 to 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-1 low-dose and high-dose groups and the Kanglaite soft capsule group was significantly reduced from D13 to D25 (P≤0.05 or P≤0.01).

[0198] As shown in Table 5, compared with the model control group, the relative tumor volumes of mice in the cisplatin group, the cisplatin combined with Kanglaite soft capsule group, and the cisplatin combined with low- and high-dose GLP-1 groups were significantly reduced from Day 13 to Day 25 (P ≤ 0.05 or P ≤ 0.01). The relative tumor volume in the Kanglaite soft capsule group was not statistically significant. Compared with the cisplatin + Kanglaite soft capsule group, the tumor volume of mice in the cisplatin combined with high-dose GLP-1 group was significantly reduced from Day 19 to Day 25 (P ≤ 0.05), while no statistical significance was observed in the other groups.

[0199] Table 4 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on tumor volume in Lewis lung cancer-bearing mice

[0200] 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 Kanglaite soft capsule group, & P≤0.05, && P≤0.01, compared with cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0201] Table 5 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the relative tumor volume in Lewis cell tumor-bearing mice

[0202] Note: Compared with the model control group, *P≤0.05, **P≤0.01.

[0203] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the relative tumor proliferation rate (T / C) in Lewis lung cancer-bearing mice

[0204] As shown in Table 6, the T / C administration rates of mice in the low-dose cisplatin + GLP-1 group from D7 to D25 were 50.0%, 21.2%, 3.5%, 3.3%, 7.2%, 11.0%, and 11.3%, respectively, and the values ​​were all ≤ 60.0%; the T / C administration rates of mice in the high-dose cisplatin + GLP-1 group from D10 to D25 were 27.3%, 0.0%, 0.6%, 0.4%, 1.4%, and 0.8%, respectively, and the values ​​were all ≤ 60.0%. All were ≤60.0%; the T / C administration rates of mice in the cisplatin group on D7-D10 and D16-D25 were 45.5%, 27.3%, 28.2%, 36.9%, 30.9% and 23.6% respectively, and the values ​​were all ≤60.0%; the T / C administration rates of mice in the cisplatin + Kanglaite soft capsule group on D13-D25 were 7.3%, 3.3%, 15.6%, 7.3% and 15.6% respectively, and the values ​​were all ≤60.0%.

[0205] Table 6 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the relative tumor growth rate in Lewis lung cancer-bearing mice

[0206] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on organ coefficients and tumor growth inhibition rate in Lewis lung cancer-bearing mice

[0207] As shown in Table 7, compared with the normal control group, the spleen and tumor coefficients in 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 in the cisplatin group, the low-dose cisplatin combined with GLP-1 group, and the Kanglaite soft capsule group were significantly decreased (P ≤ 0.05 or P ≤ 0.01). The spleen and tumor coefficients in the high-dose cisplatin combined with GLP-1 group were significantly decreased (P ≤ 0.01). Compared with the cisplatin group, the tumor coefficient in the high-dose cisplatin combined with GLP-1 group was significantly decreased (P ≤ 0.05), while the spleen and tumor coefficients in the Kanglaite soft capsule group were significantly increased (P ≤ 0.01). Compared with the Kanglaite soft capsule group, the spleen and tumor coefficients in the low- and high-dose cisplatin combined with GLP-1 groups were significantly decreased (P ≤ 0.01). The organ-to-body ratios in the groups were not statistically significant compared with the cisplatin + Kanglaite soft capsule group. The tumor growth inhibition rate of the Kanglaite soft capsule group was -6.8%, with a value of <40.0%; the tumor growth inhibition rates of mice in the cisplatin group, cisplatin combined with Kanglaite soft capsule group, and low- and high-dose GLP-1 groups were 61.6%, 78.3%, 77.6%, and 96.7%, respectively, with values ​​all ≥40.0%.

[0208] Table 7 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on organ coefficients and tumor growth inhibition rate in Lewis lung cancer-bearing mice

[0209] 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 Kanglaite soft capsule group, & P≤0.05, && P≤0.01; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0210] In Figure 20, the control groups are marked as follows: 1: model control group, 2: cisplatin group, 3: Kanglaite soft capsule group, 4: cisplatin + Kanglaite soft capsule group, 5: cisplatin + GLP-1 low-dose group, 6: cisplatin + GLP-1 high-dose group.

[0211] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on blood biochemical parameters in Lewis lung cancer-bearing mice

[0212] As shown in Table 8, compared with the normal control group, the blood WBC, ALT, AST, and BUN levels of mice in the model control group were significantly increased (P ≤ 0.05 or P ≤ 0.01), and the RBC levels were significantly decreased (P ≤ 0.05). Compared with the model control group, the blood WBC and AST levels of mice in the high-dose cisplatin combined with GLP-1 group were significantly decreased (P ≤ 0.05 or P ≤ 0.01), the blood WBC, ALT, and AST levels of mice in the low-dose cisplatin combined with GLP-1 group were significantly decreased (P ≤ 0.05), and the blood WBC levels of mice in the cisplatin group and the cisplatin combined with Kanglaite soft capsule group were significantly decreased (P ≤ 0.05). Compared with the cisplatin group, the blood RBC levels of mice in the high-dose cisplatin combined with GLP-1 group were significantly increased (P ≤ 0.05). Compared with the Kanglaite soft capsule group, the blood WBC levels of mice in the high-dose cisplatin combined with GLP-1 group were significantly decreased (P ≤ 0.05), and the blood WBC levels of mice in the cisplatin combined with Kanglaite soft capsule group were significantly decreased (P ≤ 0.05). Compared with the cisplatin + Kanglaite soft capsule group, the WBC count in the blood of mice in the cisplatin combined with low-dose GLP-1 group was significantly increased (P≤0.05), while there was no statistical significance in the other groups.

[0213] Table 8 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on blood biochemical indicators in Lewis lung cancer-bearing mice

[0214] 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 Kanglaite soft capsule group, & P≤0.05; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05.

[0215] Effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on CD3 in Lewis lung cancer-bearing mice + / CD4 + 、CD3 + / CD8 + Impact

[0216] As shown in Table 9, compared with the normal control group, the blood CD3 + / CD8 + Compared with the model control group, the CD3 + / CD8 + Compared with the Kanglaite soft capsule group, the CD3 + / CD8 + Compared with the cisplatin + Kanglaite soft capsule group, there was no statistical significance in the other drug groups.

[0217] Table 9 Effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on CD3 + / CD4 + 、CD3 + / CD8 + Effect of content

[0218] 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 Kanglaite soft capsule group, & P≤0.05, && P≤0.01; compared with the cisplatin + Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0219] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on bone marrow cells in Lewis lung cancer-bearing mice

[0220] As shown in Table 10, compared with the normal control group, the model control group showed a significant increase in the monocyte system in the bone marrow (P ≤ 0.05). The granulocyte-erythrocyte system, granulocyte-erythrocyte ratio, and other cells showed an increasing trend. The erythrocyte system and lymphocyte system showed a decreasing trend, but the trend was not statistically significant. Compared with the model control group, the erythrocyte system in the bone marrow of mice in the low-dose cisplatin combined with GLP-1 group and the Kanglaite soft capsule group was significantly increased (P ≤ 0.05), and the granulocyte-erythrocyte ratio and monocyte system were significantly decreased (P ≤ 0.05). The monocyte system in the bone marrow of mice in the Kanglaite soft capsule group and the high-dose cisplatin combined with GLP-1 group was significantly decreased (P ≤ 0.01). Compared with the cisplatin group, there was no statistically significant difference between the treatment groups. Compared with the Kanglaite soft capsule group, the bone marrow lymphocyte system in the low- and high-dose cisplatin combined with GLP-1 groups was significantly increased (P ≤ 0.05). Compared with the cisplatin + Kanglaite soft capsule group, the bone marrow granulocyte-erythroid system of mice in the cisplatin combined with GLP-1 high-dose group was significantly increased (P≤0.05) and the erythrocyte system was significantly decreased (P≤0.05), while there was no statistical significance in the other groups.

[0221] Table 10 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on bone marrow cells in Lewis lung cancer-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 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] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on tumor, spleen, kidney and stomach tissue pathology in Lewis cell tumor-bearing mice

[0224] As shown in Figure 21, the model control group showed abundant tumor cells surrounding the tumor, with densely arranged cells, large cell size, a high nuclear-cytoplasmic ratio, and frequent mitotic figures. In the cisplatin group, extensive necrosis and hemorrhage were observed in the tumor center, and the number of tumor cells was abundant. Necrosis was observed in the tumor center of the cisplatin + Kanglaite Soft Capsule group and the cisplatin + high-dose GLP-1 group, and the number of surrounding tumor cells decreased.

[0225] As shown in Figures 22 and 23, no obvious abnormalities were found in the kidney and stomach pathology of each group.

[0226] As shown in Figure 24, the model control group showed obvious red pulp hematopoiesis with diverse cell types. The cisplatin + Kanglaite soft capsule group showed no obvious abnormalities in the spleen, while the spleens of the other groups showed varying degrees of red pulp hematopoiesis with diverse cell types.

[0227] In Figure 21, the control groups are marked as follows: A: normal control group, B: model control group, C: cisplatin group; D: Kanglaite soft capsule group; E: cisplatin + Kanglaite soft capsule group; F: cisplatin + GLP-1 low-dose group, G: cisplatin + GLP-1 high-dose group

[0228] In Figures 22 and 23, the control groups are marked as follows: A: normal control group, B: model control group, C: cisplatin group; D: Kanglaite soft capsule group; E: cisplatin + Kanglaite soft capsule group; F: cisplatin + GLP-1 low-dose group, G: cisplatin + GLP-1 high-dose group

[0229] In Figure 24, the control groups are marked as follows: A: normal control group, B: model control group, C: cisplatin group; D: Kanglaite soft capsule group; E: cisplatin + Kanglaite soft capsule group; F: cisplatin + GLP-1 low-dose group, G: cisplatin + GLP-1 high-dose group

[0230] in conclusion

[0231] Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin can significantly inhibit the growth of tumors in Lewis lung cancer-bearing mice and has a significant synergistic effect.

[0232] Discussion and Summary

[0233] In recent years, the incidence of lung cancer has steadily climbed, with a high mortality rate and a trend toward younger patients, driven by environmental degradation and factors such as smoking. Clinically, the primary treatment options are surgery, radiotherapy, and chemotherapy to achieve early tumor eradication and reduce tumor burden. However, due to the high invasiveness and metastasis of lung cancer cells and their insensitivity to radiotherapy and chemotherapy, patients may experience postoperative immunosuppression, which accelerates tumor metastasis and progression, leading to a poor clinical prognosis.

[0234] The results of this experiment showed that the tumor volume of the mice in the model control group increased significantly, the thymus index decreased significantly, the spleen 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. In addition, CD3 + The content increased significantly, CD4 + Increased content, CD8 + This indicated that the lymphatic system function of the model control group mice declined during tumorigenesis.

[0235] Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin can significantly inhibit the growth of mouse tumors, significantly reduce the thymus index and spleen index, significantly reduce the number of red blood cells in the mouse blood, significantly increase the number of white blood cells, and gradually return to normal liver and kidney function indicators; CD4 + The content has decreased, CD8 + The results showed that the combined use of GLP-1 and cisplatin can protect immune organs and enhance the body's autoimmune function, thereby reducing toxicity and improving anti-tumor effects. Compared with the Kanglaite soft capsule group and the cisplatin combined with Kanglaite soft capsule group, the Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin can significantly inhibit tumor growth, and the inhibitory effect of Ganoderma lucidum polysaccharide GLP-1 on tumor growth is significantly stronger than that of Kanglaite soft capsule.

[0236] In summary, Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin can significantly inhibit the growth of tumors in Lewis lung cancer mice and has a significant synergistic effect.

[0237] Experimental study and data on the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on Lewis lung cancer-bearing mice

[0238] Study Objectives

[0239] In this experiment, Lewis lung cancer cells were inoculated in the right axilla of C57 mice to prepare tumor-bearing mice. The mechanism by which Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy increases chemotherapy tolerance was studied, providing an experimental basis for its clinical research.

[0240] Experimental Materials

[0241] Test sample

[0242] Ganoderma lucidum polysaccharide GLP-1, batch number: ALM20200518A1; provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0243] Positive control

[0244] Kanglaite soft capsules, batch number: 20210904, Zhejiang Kanglaite Pharmaceutical Co., Ltd.; cisplatin injection, batch number: 601211204, Jiangsu Hausen Pharmaceutical Group Co., Ltd.

[0245] experimental animals

[0246] 95 SPF male C57 mice, weighing 12-15 g, were provided by Hunan Slake Jingda Experimental Animal Co., Ltd., with experimental animal production license number: SCXK (Xiang) 2019-0004 and experimental animal quality certificate number: 430727211103153225. They were raised in Area D of the barrier environment animal laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd., with experimental animal use license number: SYXK (Xiang) 2020-0015.

[0247] Main reagents

[0248] 0.9% sodium chloride injection, batch number: 21071401C, product of Hunan Kangyuan Pharmaceutical Co., Ltd.; ALT assay kit, batch number: 201751, AST assay kit, batch number: 110620, CRE assay kit, batch number: 111644, BUN assay kit, batch number: 201749, all are products of Wako Pure Chemical Industries, Ltd. of Japan.

[0249] Main instruments

[0250] S10 portable high-speed disperser, Shanghai Xinzhi; AR223CN electronic balance, Ohaus Instruments (Changzhou) Co., Ltd.; LABOSPECT003 automatic biochemical analyzer, Hitachi, Japan; TDZ5-WS desktop multi-tube automatic balancing centrifuge, Hunan Kaida Industrial Development Co., Ltd.; ME2002E electronic balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.; digital display vernier caliper, Yongkang Zhengfeng Hardware Co., Ltd.; flow cytometer, BD; ASP200S fully automatic tissue dehydrator, ASP300S fully automatic tissue dehydrator, TP1020 fully automatic dehydrator, HI1210 slide spreader, HI1220 slide baking machine, RM2235 paraffin slicer, EG1150H+C tissue embedding machine, AutoStainer XL automatic slide stainer + CV5030 automatic coverslip machine, Leica, Germany; BX43 biological microscope + MD50 digital imaging system, CX31 biological microscope, Olympus, Japan.

[0251] Research content

[0252] Experimental methods

[0253] 1×10 Lewis tumor cells in logarithmic growth phase were taken 7 0.2 mL of the solution was inoculated into the right axilla of 10 normal male C57 mice. The tumor growth was observed and the mice were prepared into tumor-bearing mice. When the tumor volume of the tumor-bearing mice reached 200 mm 3 At the above time, the tumor tissue of the tumor-bearing mice was aseptically removed to prepare a tissue cell suspension, and the tumor tissue homogenate was made with cell culture medium at a ratio of 1:1 (V:V). The suspension was inoculated into the right axilla of 72 male C57 mice with an inoculation volume of 0.2 mL per mouse. The suspension was kept for 24 h until the tumor volume of the tumor-bearing mice reached 100 mm. 3At the above time, mice bearing tumors without ulceration and with good growth were selected and randomly divided into groups according to tumor volume: model control group, radiotherapy group (2Gy / d), Kanglaite soft capsule group (1404mg / kg), radiotherapy + Kanglaite soft capsule group (2Gy + 1404mg / kg), radiotherapy + low-dose GLP-1 group (2Gy + 130mg / kg), and radiotherapy + high-dose GLP-1 group (2Gy + 1170mg / kg), with 10 mice in each group. In addition, 10 mice were selected as the normal control group. Except for the normal control group, the remaining animals underwent radiotherapy. The radiotherapy animals were irradiated using an animal radiotherapy device. The animals were anesthetized, placed in a homemade lead suit, and the tumor tissue was exposed for radiotherapy. The radiation intensity was 2Gy / day, and the radiation was continued for 5 consecutive days. The mice in the normal control group, model control group, and radiotherapy group were gavaged with distilled water, the mice in the chemotherapy group were intraperitoneally injected with cisplatin, and the mice in the other groups were given the corresponding drug solution, with a volume of 20 mL / kg (gavage), once a day, for 14 consecutive days. After the last dose, orbital blood was collected to detect the levels of WBC, RBC, liver and kidney function indicators (ALT, AST, CRE, BUN), and the CD3 + 、CD4 + 、CD8 + The spleen, thymus, and tumor were weighed and the organ-to-body ratio was calculated. The spleen, thymus, stomach, kidney, and nose were also taken for histopathological examination.

[0254] Dosage design

[0255] Ganoderma lucidum polysaccharide GLP-1 was designed based on the results of previous studies. The low dose was 130 mg / kg and the high dose was 1170 mg / kg. The corresponding drugs were administered as shown in Table 11. This experiment was conducted at the same multiple of the intended clinical dose.

[0256] The proposed clinical dosage of Kanglaite Soft Capsules is 0.45g / capsule, 6 capsules / time, 4 times / day, which is 10.8g / day. The equivalent dose in mice based on body surface area is 10.8g / day*0.0026 / 0.02kg=1404mg / kg. This trial was conducted at the same multiple of the proposed clinical dosage.

[0257] This study designed radiotherapy intensity to be 2 Gy / day, which is equal to the intended clinical dose.

[0258] Table 11 Trial grouping and dosage design

[0259] Detection indicators

[0260] Efficacy indicators

[0261] Evaluation of animal quality of life and survival time: During the experiment, the weight of each group of animals, animal deaths, the mental state of the mice, fur color, fur smoothness, stool form, and whether there is diarrhea are recorded in detail.

[0262] Tumor volume detection: The length and width of the mouse tumor were measured with a vernier caliper every 3 days after administration, and the tumor volume was calculated. Tumor volume (TV) = 1 / 2 × a (length) × b 2 (width). Relative tumor volume (RTV) = TV t / TV0, TV0 is the tumor volume at the time of drug administration, TV t is the tumor volume measured each time. Relative tumor proliferation rate T / C (%) = T RTV / C RTV ×100%,T RTV :RTV in treatment group; C RTV : Model control group RTV.

[0263] Immune Organs: After the final dose, the spleen and thymus were weighed and the organ coefficient was calculated. All animal parameters were photographed and recorded. Organ coefficient (%) = organ weight / fasting body weight × 100%.

[0264] Hematological examination: blood routine (WBC, RBC) and biochemical (liver and kidney function) indexes were tested after the last dose.

[0265] Histopathological examination: After the last administration, the thymus, spleen, nasal tissue, stomach, and kidney were collected for HE staining, sectioning, and embedding for pathological examination.

[0266] CD4 + 、CD8 + Content detection: After the last administration, blood lymphocyte typing CD3 was detected by flow cytometry + / CD4 + , CD3 + / CD8 + content.

[0267] Data processing and statistical analysis

[0268] The significant figures of the experimental data were rounded off and statistical analysis was performed in accordance with the SOP of this center. The statistical software used was SPSS. The measurement data were expressed as mean ± standard deviation. Normality and homogeneity of variance were tested using the Leven's test. If there was no statistical significance (P>0.05), one-way analysis of variance (ANOVA) was used for statistical analysis. If the ANOVA was statistically significant (P≤0.05), the LSD test (parametric method) was used for comparative analysis. If the variances were unequal (P≤0.05), the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P≤0.05), the Dunnett's test (nonparametric method) was used for comparative analysis. Statistical results were tested with an α=0.05 threshold, where P≤0.05 indicated statistical significance and P≤0.01 indicated that the difference being tested was highly significant.

[0269] Experimental results

[0270] General clinical observations and animal deaths

[0271] Before administration, the mice were active, with normal movements and gait. Following administration, the radiotherapy mice lost weight. Mice 3M01, 3M10, 3M03, and 3M02 in the radiotherapy group died on Day 10, Day 11, and Day 12, respectively. Mice 6M01 and 6M10 in the radiotherapy combined with Kanglaite soft capsule group died on Day 10. Mice 7M05 in the radiotherapy combined with low-dose GLP-1 group died on Day 7. Mice 8M08 and 8M05 in the radiotherapy combined with high-dose GLP-1 group died on Day 7 and Day 10, respectively.

[0272] The mortality rates of each group were 0%, 0%, 40%, 0%, 20%, 10%, and 20%, respectively.

[0273] Table 12 Statistics of the number of surviving animals and mortality rates in each group

[0274] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on body weight in Lewis cell tumor-bearing mice

[0275] As shown in Table 13, compared with the normal control group, the body weight of mice in the model control group was significantly reduced on day 1 after administration (P ≤ 0.01). Compared with the model control group, the body weight of mice in the radiotherapy combined with low-dose and high-dose GLP-1 groups and the Kanglaite soft capsule group was significantly reduced on day 4 after administration; the body weight of mice in the radiotherapy combined with low-dose and high-dose GLP-1 groups and the Kanglaite soft capsule group was significantly reduced on day 7 after administration (P ≤ 0.01); the body weight of mice in the radiotherapy combined with low-dose and high-dose GLP-1 groups and the Kanglaite soft capsule group was significantly reduced on day 10 after administration (P ≤ 0.05 or P ≤ 0.01); the body weight of mice in the radiotherapy combined with low-dose and high-dose GLP-1 groups and the Kanglaite soft capsule group was significantly reduced on day 13 after administration (P ≤ 0.05 or P ≤ 0.01). Compared with the radiotherapy group, the body weight of mice in the radiotherapy combined with low-dose and high-dose GLP-1 groups, and the Kanglaite soft capsule group was significantly reduced on day 4 (P≤0.05 or P≤0.01). The body weight of mice in the Kanglaite soft capsule group was significantly increased on day 7 to day 10 (P≤0.05 or P≤0.01). The body weight of mice in the radiotherapy combined with Kanglaite soft capsule group was significantly reduced on day 10 to day 13 (P≤0.05). The body weight of mice in the radiotherapy combined with low-dose GLP-1 group was significantly reduced on day 13 (P≤0.05 or P≤0.01). Compared with the Kanglaite soft capsule group, the body weight of mice in the radiotherapy combined with low-dose GLP-1 group and the Kanglaite soft capsule group was significantly reduced on day 4 (P≤0.05 or P≤0.01). The body weight of mice in the radiotherapy combined with low-dose GLP-1 group and the Kanglaite soft capsule group was significantly reduced on day 7 to day 13 (P≤0.01). Compared with the radiotherapy combined with Kanglaite soft capsule group, there was no statistical difference among the groups.

[0276] Table 13 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on the body weight of Lewis cell tumor-bearing mice (Mean ± SEM)

[0277] Note: Compared with the normal control group, ++ P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01; compared with the radiotherapy group, # P≤0.05, ## P≤0.01; compared with Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01; compared with radiotherapy + Kanglaite soft capsule group, ■ P≤0.05, ■■ P≤0.01

[0278] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on tumor volume and relative tumor volume in Lewis cell tumor-bearing mice

[0279] As shown in Table 14, compared with the model control group, the tumor volume of mice in the radiotherapy combined with low-dose GLP-1 and Kanglaite soft capsule groups was significantly reduced from Day 7 to Day 13 (P ≤ 0.05 or P ≤ 0.01). The tumor volume of mice in the radiotherapy combined with high-dose GLP-1 group was significantly reduced from Day 13 (P ≤ 0.05 or P ≤ 0.01). The tumor volume of mice in the radiotherapy group was significantly reduced from Day 7 to Day 13 (P ≤ 0.01). Compared with the radiotherapy group, the tumor volume of mice in the Kanglaite soft capsule group was significantly increased from Day 10 (P ≤ 0.01). Compared with the Kanglaite soft capsule group, the tumor volume of mice in the radiotherapy combined with low-dose GLP-1 and Kanglaite soft capsule groups was significantly reduced from Day 7 to Day 13 (P ≤ 0.05 or P ≤ 0.01). The tumor volume of mice in the radiotherapy combined with high-dose GLP-1 group was significantly reduced from Day 7 (P ≤ 0.05).

[0280] As shown in Table 15, compared with the model control group, the relative tumor volumes of mice in the radiotherapy combined with Kanglaite soft capsule group and the low-dose GLP-1 group were significantly reduced from Day 7 to Day 13 after administration (P ≤ 0.05 or P ≤ 0.01); the relative tumor volumes of mice in the radiotherapy group were significantly reduced from Day 7 after administration (P ≤ 0.05). Compared with the Kanglaite soft capsule group, the relative tumor volumes of mice in the radiotherapy combined with low-dose GLP-1 group and the Kanglaite soft capsule group were significantly reduced from Day 7 to Day 13 after administration (P ≤ 0.05 or P ≤ 0.01).

[0281] Table 14 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on tumor volume in Lewis cell-bearing mice (Mean ± SEM)

[0282] Note: Compared with the model control group, *P≤0.05, **P≤0.01; compared with the radiotherapy group, # P≤0.05, ## P≤0.01; compared with Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01.

[0283] Table 15 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on the relative tumor volume of Lewis cell tumor-bearing mice (Mean ± SEM)

[0284] Note: Compared with the model control group, *P≤0.05, **P≤0.01; compared with the radiotherapy group, # P≤0.05; compared with radiotherapy + cisplatin group, & P≤0.05, && P≤0.01; compared with Kanglaite soft capsule group, ★ P≤0.05, ★★P≤0.01.

[0285] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on the relative tumor proliferation rate (T / C) in Lewis cell tumor-bearing mice

[0286] As shown in Table 16, the T / C of mice in the radiotherapy combined with Kanglaite soft capsule group on days 7 to 13 after administration were 47.0%, 9.0%, and 13.2%, respectively, all ≤ 60.0%; the T / C of mice in the radiotherapy combined with low-dose GLP-1 group on days 7 to 13 after administration were 22.1%, 25.3%, and 18.3%, respectively, all ≤ 60.0%.

[0287] Table 16 Effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the relative tumor growth rate of mice (Mean ± SEM)

[0288] Note: Relative tumor growth rate: T / C (%)>60 is ineffective; T / C (%) ≤60 is effective.

[0289] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on organ coefficients and tumor growth inhibition rate in Lewis cell tumor-bearing mice

[0290] As shown in Table 17, compared with the normal control group, the organ-to-body ratios of the spleen and tumor tissues in the model control group were significantly increased (P ≤ 0.01), while the organ-to-body ratio of the thymus was significantly decreased (P ≤ 0.05). Compared with the model control group, the spleen coefficient in the radiotherapy group was significantly decreased (P ≤ 0.01); the spleen coefficient in the radiotherapy combined with high-dose GLP-1 group was significantly decreased (P ≤ 0.05); both the spleen coefficient and tumor coefficient in the radiotherapy combined with low-dose GLP-1 group were significantly decreased (P ≤ 0.05 or P ≤ 0.01); and the thymus coefficient and spleen coefficient in the radiotherapy combined with Kanglaite soft capsule group were significantly decreased (P ≤ 0.01). Compared with the radiotherapy group, the thymus index in the Kanglaite soft capsule group was significantly increased (P ≤ 0.01). Compared with the Kanglaite soft capsule group, the spleen coefficient and tumor coefficient in the radiotherapy combined with low-dose GLP-1 group were significantly decreased (P ≤ 0.01). No statistical differences were found among the other groups. The tumor growth inhibition rates of each group were: 25.0%, -35.9%, 67.6%, 75.5%, and -8.6%, respectively.

[0291] Table 17 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on organ coefficients and tumor growth inhibition rate in Lewis cell tumor-bearing mice (Mean ± SEM)

[0292] Note: Compared with the normal control group, + P≤0.05, ++ P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01; compared with the radiotherapy group,# P≤0.05, ## P≤0.01; compared with Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01; compared with radiotherapy + Kanglaite soft capsule group, ■ P≤0.05, ■■ P≤0.01.

[0293] As shown in Figure 25, the corresponding labels are: 2: model control group, 3: radiotherapy group, 5: Kanglaite soft capsule group, 6: radiotherapy + Kanglaite soft capsule group; 7: radiotherapy + GLP-1 low-dose group, 8: radiotherapy + GLP-1 high-dose group

[0294] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on blood biochemical parameters in Lewis cell tumor-bearing mice

[0295] As shown in Table 18, compared with the normal control group, the WBC, ALT, AST, and BUN of the model control group were significantly increased (P≤0.05 or P≤0.01), and the RBC was significantly decreased (P≤0.05). Compared with the model control group, the WBC of mice in the radiotherapy combined with low-dose and high-dose GLP-1 groups, and the Kanglaite soft capsule group were significantly decreased after administration (P≤0.05 or P≤0.01); the RBC of mice in the radiotherapy combined with low-dose GLP-1 group was significantly increased after administration (P≤0.05 or P≤0.01); the ALT of mice in the Kanglaite soft capsule group was significantly decreased after administration (P≤0.05 or P≤0.01); the BUN of mice in the radiotherapy combined with low-dose and high-dose GLP-1 groups, and the Kanglaite soft capsule group were significantly decreased after administration (P≤0.05); and the CRE of mice in the radiotherapy combined with Kanglaite soft capsule group was significantly decreased after administration (P≤0.05). Compared with the radiotherapy group, the white blood cell count (WBC) count of mice in the radiotherapy combined with low- and high-dose GLP-1 groups, as well as the Kanglaite soft capsule group, was significantly decreased after administration (P ≤ 0.05 or P ≤ 0.01). The red blood cell count (RBC) count of mice in the radiotherapy combined with low-dose GLP-1 group was significantly increased after administration (P ≤ 0.05 or P ≤ 0.01). The creatinine erythrocyte count (CRE) count of mice in the radiotherapy combined with Kanglaite soft capsule group was significantly decreased after administration (P ≤ 0.05). Compared with the Kanglaite soft capsule group, the white blood cell count (WBC) count of mice in the radiotherapy combined with low- and high-dose GLP-1 groups, as well as the Kanglaite soft capsule group, was significantly decreased after administration (P ≤ 0.05 or P ≤ 0.01). The AST count of mice in the radiotherapy combined with low-dose GLP-1 group and the Kanglaite soft capsule group was significantly decreased after administration (P ≤ 0.05 or P ≤ 0.01). There were no statistically significant differences between the groups compared with the radiotherapy combined with Kanglaite soft capsule group.

[0296] Table 18 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on blood biochemical indicators in Lewis cell tumor-bearing mice (Mean ± SEM)

[0297] Note: Compared with the normal control group, + P≤0.05, ++ P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01; compared with the radiotherapy group, # P≤0.05, ## P≤0.01; compared with Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01; compared with radiotherapy + Kanglaite soft capsule group, ■ P≤0.05.

[0298] Effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on CD3 in Lewis cell tumor-bearing mice + / CD4 + 、CD3 + / CD8 + Effect of content

[0299] As shown in Table 19, compared with the normal control group, the CD3 + / CD8 + Compared with the model control group, the CD3 + / CD4 + The CD3 expression of mice in the radiotherapy combined with Kanglaite soft capsule group was significantly decreased (P≤0.05). + / CD8 + Compared with the radiotherapy group, the CD3 + / CD4 + The CD3 expression of mice in the radiotherapy combined with Kanglaite soft capsule group was significantly decreased (P≤0.05). + / CD8 + The difference in the other groups was not significant (P≤0.05).

[0300] Table 19 Effect of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on CD3 + / CD4 + 、CD3 + / CD8 + Effect of content (Mean±SEM)

[0301] Note: Compared with the normal control group, + P≤0.05; compared with the model control group, *P≤0.05; compared with the radiotherapy group, # P≤0.05,## P≤0.01; compared with Kanglaite soft capsule group, ★ P≤0.05, ★★ P≤0.01; compared with radiotherapy + Kanglaite soft capsule group, ■ P≤0.05.

[0302] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy on the pathological changes of thymus, spleen, nose, stomach and kidney tissues in Lewis cell tumor-bearing mice

[0303] As shown in Figures 26, 27, 28, 29, and 30, mice in the model control group had diffuse red pulp and increased extramedullary lesions in the spleen. Mice in the radiotherapy group had decreased thymic lymphocytes and diffuse medullary lesions; increased extramedullary hematopoiesis in the red pulp and decreased white pulp lymphocytes in the spleen; and multiple hemorrhages in the nasal concha propria. Following Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy, the number of thymic medullary lymphocytes decreased, the number of plasma cells in the white pulp of the spleen increased, and no abnormal lesions were observed in the nasal concha. No abnormal lesions were observed in the stomach or kidneys in any of the treatment groups.

[0304] As shown in Figures 26, 27, 28, 29, and 30, the controls are marked as: A: normal control group, B: model control group, C: radiotherapy group; E: Kanglaite soft capsule group; F: radiotherapy + Kanglaite soft capsule group; G: radiotherapy + low-dose GLP-1 group, H: radiotherapy + high-dose GLP-1 group.

[0305] in conclusion

[0306] Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy can significantly inhibit the growth of tumors in Lewis lung cancer-bearing mice and has a significant synergistic effect.

[0307] Discussion and Summary

[0308] In recent years, the incidence of lung cancer has steadily climbed, with a high mortality rate and a trend toward younger patients, driven by environmental degradation and factors such as smoking. Clinically, the primary treatment options are surgery, radiotherapy, and chemotherapy to achieve early tumor eradication and reduce tumor burden. However, due to the high invasiveness and metastasis of lung cancer cells and their insensitivity to radiotherapy and chemotherapy, patients may experience postoperative immunosuppression, which accelerates tumor metastasis and progression, leading to a poor clinical prognosis.

[0309] The results of this experiment showed that the tumor volume of the mice in the model control group increased significantly, the thymus index decreased significantly, the spleen 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. In addition, the CD4 + Increased content, CD8 +Significantly decreased, indicating that the lymphatic system function of mice in the model control group decreased during tumor development. Tumor volume and body weight were significantly reduced in mice in the radiotherapy group, and the mortality rate was 40%. The results of this experiment show that GLP-1, a polysaccharide from Ganoderma lucidum, combined with radiotherapy can significantly inhibit tumor growth in mice. Thymic and spleen indices were significantly reduced, red blood cell counts were significantly decreased, white blood cell counts were significantly increased, and liver and kidney function indicators were reduced. Histopathological results showed that GLP-1 increased the number of plasma cells in the splenic white pulp. These results suggest that the combination of GLP-1 and radiotherapy can protect immune organs and enhance the body's autoimmune function, thereby reducing toxicity and enhancing anti-tumor effects. Compared with the Kanglaite soft capsule group and the radiotherapy combined with Kanglaite soft capsule group, the GLP-1 polysaccharide combined with radiotherapy significantly inhibited tumor growth, and the inhibitory effect of GLP-1 on tumor growth was significantly stronger than that of Kanglaite soft capsule.

[0310] In summary, Ganoderma lucidum polysaccharide GLP-1 combined with radiotherapy can significantly inhibit the growth of tumors in Lewis lung cancer-bearing mice and has a significant synergistic effect.

[0311] Experimental study and data on the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on H22 liver cancer-bearing mice

[0312] Purpose of the experiment

[0313] Tumor-bearing mice were prepared by inoculating H22 liver cancer tumor homogenate in the right axilla of C57 mice to study the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-1 on liver cancer-bearing mice, providing experimental basis for its clinical research.

[0314] Experimental Materials

[0315] Test sample

[0316] Ganoderma lucidum polysaccharide GLP-1, batch number: H2202208151A1; provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0317] Positive control

[0318] Cisplatin, batch number: E21268081, product of Shanghai Aladdin Biochemical Technology Co., Ltd.

[0319] experimental animals

[0320] Eighty SPF male C57 mice, weighing 14–16 g, were provided by the Guangdong Medical Laboratory Animal Center. The laboratory animal production license number is SCXK (Yue) 2022-0002, and the laboratory animal quality certificate number is 44007200107158.

[0321] Main reagents

[0322] PBS buffer was prepared by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.; fetal bovine serum was a product of Zhejiang Tianhang Biotechnology Co., Ltd.; 1640 culture medium was a product of Gibco; and 0.25% trypsin was a product of Gibco.

[0323] Main instruments

[0324] Vernier caliper, product of Shanghai Tool Factory Co., Ltd.; I-2000 balance, product of Dongguan Nancheng Changxie Electronic Products Factory; ophthalmic scissors and tweezers, product of Shanghai Jinzhong Medical Instrument Co., Ltd.; CCL-170B-8 carbon dioxide incubator, product of Singapore ESCO Company; Luna-Ⅱ cell counter, product of Nanjing Hengqiao Instrument Co., Ltd.

[0325] Experimental methods

[0326] A solid tumor model was established by subcutaneously injecting a suspension of H22 liver cancer solid tumors into the axilla of 65 healthy male C57 mice. When the average tumor volume of all mice reached approximately 230 mm³, the mice were randomly divided into groups based on tumor volume and given the corresponding drug or drug vehicle by gavage or intraperitoneal injection for 25 consecutive days. Tumor length and short diameter were measured every 3 days to calculate tumor volume, and mice were weighed every 3 days. At the end of the experiment, the tumors, spleen, and thymus of the mice were weighed, and the tumor index, spleen index, and thymus index were calculated.

[0327] Dosage design

[0328] Ganoderma lucidum polysaccharide GLP-1 was designed based on previous experimental results. The low dose was 50 mg / kg and the high dose was 150 mg / kg. The corresponding drugs were administered as shown in Table 20.

[0329] Cisplatin dosage design basis: According to the clinical dosage of cisplatin, each person should not exceed 100 mg / m2 per day. 2 The tolerance of mice to cisplatin was evaluated and a dose of 3 mg / kg was selected as the administration dose.

[0330] Table 20 Trial grouping and dosage design

[0331] Detection indicators

[0332] Efficacy indicators

[0333] Relative tumor inhibition rate

[0334] Relative tumor inhibition rate (%) = (1-T RTV / C RTV )×100%. Among them, T RTV is the relative tumor volume of the experimental group, C RTV is the relative tumor volume of the model control group.t / V0,V t =V0 is the tumor volume of mice on day t of drug administration, and V0 is the tumor volume of mice at the time of grouping. Evaluation criteria: Relative tumor inhibition rate ≥ 40%, and P < 0.05 after statistical analysis is considered an effective inhibition level.

[0335] Tumor growth inhibition rate

[0336] Tumor growth inhibition rate (%) = (1-T / C) × 100%. Where T represents the average tumor weight of the treatment group and C represents the average tumor weight of the model control group. Evaluation criteria: Tumor growth inhibition rate ≥ 40% and statistical analysis P < 0.05 are considered effective inhibition levels.

[0337] Spleen and thymus organ coefficients: After the last administration, the spleen, thymus, and tumor were weighed and the organ coefficients were calculated.

[0338] Tumor index (%) = tumor weight / body weight × 100%.

[0339] Immune organ index (mg / g) = organ mass / body weight × 1000.

[0340] Data processing and statistical analysis

[0341] SPSS 17.0 was used for statistical analysis, and the statistical significance level was set at P ≤ 0.05. The measurement data were expressed as mean ± standard deviation. Leven's test was used to test normality and homogeneity of variance. If normality and homogeneity of variance were met (P>0.05), one-way analysis of variance (ANOVA) and LSD test were used for statistical analysis. If normality and homogeneity of variance were not met (P<0.05), Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P<0.05), Dunnett's Test (non-parametric method) was used for comparative analysis. Statistical differences and biological significance were considered in the evaluation.

[0342] Experimental results

[0343] Animal deaths

[0344] As shown in Table 21, the mortality rate of mice in each group was 0.

[0345] Table 21 Statistics of the number of surviving animals and mortality rates in each group

[0346] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on body weight in H22 liver cancer-bearing mice

[0347] As shown in Table 22, compared with the normal group, the model control group had significantly increased body weight from D21 to D25, the cisplatin group had significantly decreased body weight from D18 to D25, and the cisplatin + GLP-1 low-dose group and the cisplatin + GLP-1 high-dose group had significantly decreased body weight from D15 to D25. Compared with the model control group, the cisplatin group and the cisplatin + GLP-1 low-dose group had significantly decreased body weight from D9 to D25; the cisplatin + GLP-1 high-dose group had significantly decreased body weight from D6 to D25. Compared with the cisplatin group, the cisplatin + GLP-1 high-dose group had significantly decreased body weight from D25 to D25.

[0348] [Corrected 27.03.2024 according to Rule 91] Table 22 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the body weight of H22 liver cancer-bearing mice 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.

[0349] 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.

[0350] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on tumor volume in H22 liver cancer-bearing mice

[0351] As shown in Table 23, compared with the model control group, the tumor volumes of the cisplatin group, the cisplatin + GLP-1 low-dose group, and the cisplatin + GLP-1 high-dose group were significantly reduced from D3 to D25.

[0352] Compared with the cisplatin group, the tumor volume of the low-dose cisplatin + GLP-1 group and the high-dose cisplatin + GLP-1 group was significantly reduced from D15 to D25.

[0353] Table 23 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on tumor volume in H22 liver cancer-bearing mice

[0354] Note: Compared with the model control group, * P<0.05; compared with the cisplatin group, # P<0.05.

[0355] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on relative tumor inhibition rate in H22 liver cancer-bearing mice

[0356] As shown in Table 24, compared with the model control group, the relative tumor inhibition rate in the cisplatin group was over 60% starting on Day 12, reaching a peak of 86.6%. The relative tumor inhibition rates in the low-dose and high-dose cisplatin + GLP-1 groups were over 40% starting on Day 9, reaching a peak of 94.3%. Compared with the cisplatin group, the relative tumor inhibition rates in the low-dose cisplatin + GLP-1 group ranged from 30.4% to 54.8% from Day 15 to Day 25 (P < 0.05), and the relative tumor inhibition rates in the high-dose cisplatin + GLP-1 group ranged from 52.4% to 69.0% from Day 15 to Day 25 (P < 0.05).

[0357] Table 24 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the relative tumor inhibition rate of H22 liver cancer-bearing mice (compared with the model control group)

[0358] Note: Compared with the model control group, * P<0.05.

[0359] Table 25 Effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the relative tumor inhibition rate of H22 liver cancer-bearing mice (compared with the cisplatin group)

[0360] Note: Compared with cisplatin group, # P<0.05.

[0361] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on organ coefficients and tumor growth inhibition rate in H22 liver cancer-bearing mice

[0362] As shown in Table 26, compared with the model group, the tumor index, spleen index, and thymus index were significantly reduced in the cisplatin group and the cisplatin combined with GLP-1 group. Compared with the cisplatin group, the tumor index was significantly reduced in the high-dose cisplatin + GLP-1 group, while there were no significant differences in the spleen index and thymus index in the cisplatin combined with GLP-1 group. Compared with the normal group, the thymus index was significantly reduced in the cisplatin combined with GLP-1 group.

[0363] Compared with the model control group, the tumor growth inhibition rates in the cisplatin group were 84.2%, and those in the low-dose cisplatin + GLP-1 group and the high-dose cisplatin + GLP-1 group were 88.2% and 92.8%, respectively. Compared with the cisplatin group, the tumor growth inhibition rates in the low-dose cisplatin + GLP-1 group and the high-dose cisplatin + GLP-1 group were 25.3% and 54.4%, respectively.

[0364] Table 26 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on organ index and tumor growth inhibition rate in H22 liver cancer-bearing mice

[0365] 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.

[0366] Figures 31, 32, 33, and 34 are pictures of tumor-bearing mice, and the corresponding groups are: Figure 31: Model control group, Figure 32: Cisplatin group, Figure 33: Cisplatin + GLP-1 low-dose group, Figure 34: Cisplatin + GLP-1 high-dose group

[0367] Figures 35, 36, 37, and 38 show tumor images of tumor-bearing mice, and the corresponding groups are: Figure 35: Model control group, Figure 36: Cisplatin group, Figure 37: Cisplatin + GLP-1 low-dose group, Figure 38: Cisplatin + GLP-1 high-dose group

[0368] in conclusion

[0369] Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin can significantly inhibit the growth of tumors in H22 liver cancer-bearing mice and has a significant synergistic effect.

[0370] Experimental study and data on the anti-tumor effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on 4T1 breast cancer-bearing mice

[0371] Purpose of the experiment

[0372] BALB / c mice were subcutaneously inoculated with 4T1 breast cancer cells on their left shoulder to prepare tumor-bearing mice. The anti-tumor effect of Ganoderma lucidum polysaccharide on breast cancer-bearing mice was studied to provide an experimental basis for its clinical research.

[0373] Experimental Materials

[0374] Test sample

[0375] Ganoderma lucidum polysaccharide GLP-1, batch number: H20220706A; provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0376] Positive control

[0377] Cisplatin, batch number: E21268081, product of Shanghai Aladdin Biochemical Technology Co., Ltd.

[0378] experimental animals

[0379] Seventy SPF female BALB / c mice, weighing 14–16 g, were provided by the Guangdong Medical Laboratory Animal Center. The laboratory animal production license number is SCXK (Guangdong) 2022-0002, and the laboratory animal quality certificate number is 44007200113250.

[0380] Main reagents

[0381] PBS buffer was prepared by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.; fetal bovine serum was a product of Zhejiang Tianhang Biotechnology Co., Ltd.; 1640 culture medium was a product of Gibco; and 0.25% trypsin was a product of Gibco.

[0382] Main instruments

[0383] Vernier caliper, product of Shanghai Tool Factory Co., Ltd.; I-2000 balance, product of Dongguan Nancheng Changxie Electronic Products Factory; ophthalmic scissors and tweezers, product of Shanghai Jinzhong Medical Instrument Co., Ltd.; CCL-170B-8 carbon dioxide incubator, product of Singapore ESCO Company; Luna-Ⅱ cell counter, product of Nanjing Hengqiao Instrument Co., Ltd.

[0384] Experimental methods

[0385] A solid tumor model was established by subcutaneously injecting a suspension of 4T1 breast cancer cells into the left dorsal region of the shoulder of 60 healthy female BALB / c mice. When the average tumor volume reached approximately 190 mm³, the mice were randomly divided into groups based on tumor volume and given the corresponding drug or vehicle by gavage or intraperitoneal injection for 18 consecutive days. Tumor diameters were measured every three days to calculate tumor volume, and mice were weighed every three days. At the end of the experiment, tumors, spleens, and thymuses were weighed, and the tumor index, spleen index, and thymus index were calculated.

[0386] Dosage design

[0387] Ganoderma lucidum polysaccharide GLP-1 was designed based on previous experimental results. The low dose was 50 mg / kg and the high dose was 150 mg / kg. The corresponding drugs were administered as shown in Table 27.

[0388] Cisplatin dosage design basis: According to the clinical dosage of cisplatin, each person should not exceed 100 mg / m2 per day. 2 The tolerance of mice to cisplatin was evaluated, and a dose of 4 mg / kg was selected as the administration dose.

[0389] Table 27 Trial grouping and dosage design

[0390] Detection indicators

[0391] Efficacy indicators

[0392] Relative tumor inhibition rate

[0393] Relative tumor inhibition rate (%) = (1-T RTV / C RTV )×100%. Among them, T RTV is the relative tumor volume of the experimental group, C RTVis the relative tumor volume of the model control group. t / V0,V t =V0 is the tumor volume of mice on day t of drug administration, and V0 is the tumor volume of mice at the time of grouping. Evaluation criteria: Relative tumor inhibition rate ≥ 40%, and P < 0.05 after statistical analysis is considered an effective inhibition level.

[0394] Tumor growth inhibition rate

[0395] Tumor growth inhibition rate (%) = (1-T / C) × 100%. Where T represents the average tumor weight of the treatment group and C represents the average tumor weight of the model control group. Evaluation criteria: Tumor growth inhibition rate ≥ 40% and statistical analysis P < 0.05 are considered effective inhibition levels.

[0396] Spleen and thymus organ coefficients: After the last administration, the spleen, thymus, and tumor were weighed and the organ coefficients were calculated.

[0397] Tumor index (%) = tumor weight / body weight × 100%.

[0398] Immune organ index (mg / g) = organ mass / body weight × 1000.

[0399] Data processing and statistical analysis

[0400] SPSS 17.0 was used for statistical analysis, and the statistical significance level was set at P ≤ 0.05. The measurement data were expressed as mean ± standard deviation. Leven's test was used to test normality and homogeneity of variance. If normality and homogeneity of variance were met (P>0.05), one-way analysis of variance (ANOVA) and LSD test were used for statistical analysis. If normality and homogeneity of variance were not met (P<0.05), Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P<0.05), Dunnett's Test (non-parametric method) was used for comparative analysis. Statistical differences and biological significance were considered in the evaluation.

[0401] Experimental results

[0402] Animal deaths

[0403] As shown in Table 28, the mortality rates of mice in the model control group, cisplatin group, and cisplatin + GLP-1 low-dose group were all 12.5%, and the mortality rates of mice in the other groups were 0.

[0404] Table 28 Statistics of the number of surviving animals and mortality rates in each group

[0405] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on body weight in 4T1 breast cancer-bearing mice

[0406] As shown in Table 29, compared with the normal group, the body weight of mice in the model control group increased significantly from D6 to D18, the body weight of mice in the low-dose cisplatin + GLP-1 group decreased significantly from D6 to D18, and the body weight of mice in the cisplatin group and the high-dose cisplatin + GLP-1 group decreased significantly from D3 to D18. Compared with the model control group, the body weight of mice in the cisplatin group, the low-dose cisplatin + GLP-1 group, and the high-dose cisplatin + GLP-1 group decreased significantly from D3 to D18. Compared with the cisplatin group, the body weight of mice in the high-dose cisplatin + GLP-1 group decreased significantly on D6.

[0407] Table 29 Effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the body weight of 4T1 breast cancer tumor-bearing mice

[0408] 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.

[0409] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on tumor volume in 4T1 breast cancer-bearing mice

[0410] As shown in Table 30, compared with the model control group, the tumor volumes of the cisplatin group, the cisplatin + GLP-1 low-dose group, and the cisplatin + GLP-1 high-dose group were significantly reduced from D3 to D18.

[0411] Compared with the cisplatin group, the tumor volume of the low-dose cisplatin + GLP-1 group was significantly reduced from D3 to D12, and the tumor volume of the high-dose cisplatin + GLP-1 group was significantly reduced from D6 to D18.

[0412] Table 30 Effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on tumor volume in 4T1 breast cancer-bearing mice

[0413] Note: Compared with the model control group, * P<0.05; compared with the cisplatin group, # P<0.05.

[0414] Effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the relative tumor inhibition rate of 4T1 breast cancer-bearing mice

[0415] As shown in Table 31, compared with the model control group, the relative tumor inhibition rate of the cisplatin group reached more than 40% from the 9th day, and reached a maximum of 62.8% on the 18th day. The relative tumor inhibition rate of the cisplatin + GLP-1 low-dose group was more than 40% from D3 to D18, and the relative tumor inhibition rate of the cisplatin + GLP-1 high-dose group was more than 55% from D6 to D18, with the highest being 80.0% on D18.

[0416] Compared with the cisplatin group, the relative tumor inhibition rate of the low-dose cisplatin + GLP-1 group was 23% to 35% on D3 to D12 (P<0.05), and the relative tumor inhibition rate of the high-dose cisplatin + GLP-1 group was 37% to 53% on D6 to D18 (P<0.05).

[0417] Table 31 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the relative tumor inhibition rate of 4T1 breast cancer-bearing mice (compared with the model control group)

[0418] Note: Compared with the model control group, * P<0.05.

[0419] Table 32 Effect of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on the relative tumor inhibition rate of 4T1 breast cancer-bearing mice (compared with the cisplatin group)

[0420] Note: Compared with cisplatin group, # P<0.05.

[0421] Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on organ coefficients and tumor growth inhibition rate in 4T1 breast cancer-bearing mice

[0422] As shown in Table 33, compared with the model group, the tumor index, spleen index, and thymus index were significantly reduced in the cisplatin group and the cisplatin combined with GLP-1 group. Compared with the cisplatin group, the tumor index was significantly reduced in the high-dose cisplatin + GLP-1 group, while there were no significant differences in the spleen index and thymus index in the cisplatin combined with GLP-1 group. Compared with the normal group, the thymus index was significantly reduced in the model control group and the cisplatin combined with GLP-1 group, while the spleen index was significantly increased in the model control group.

[0423] Compared with the model control group, the tumor growth inhibition rates in the cisplatin group were 71.6%, 74.1% in the low-dose cisplatin + GLP-1 group, and 82.9% in the high-dose cisplatin + GLP-1 group. Compared with the cisplatin group, the tumor growth inhibition rates in the low-dose cisplatin + GLP-1 group and high-dose cisplatin + GLP-1 group were 8.6% and 39.7%, respectively.

[0424] Table 33 Effects of Ganoderma lucidum polysaccharide GLP-1 combined with chemotherapy on organ index and tumor growth inhibition rate in 4T1 breast cancer-bearing mice

[0425] 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.

[0426] As shown in Figures 39, 40, 41, and 42, these are pictures of tumor-bearing mice, and the corresponding groups are: Figure 39: Model control group, Figure 40: Cisplatin group, Figure 41: Cisplatin + GLP-1 low-dose group, Figure 42: Cisplatin + GLP-1 high-dose group

[0427] Figures 43, 44, 45, and 46 show tumor images of tumor-bearing mice, and the corresponding groups are: Figure 43: Model control group, Figure 44: Cisplatin group, Figure 45: Cisplatin + GLP-1 low-dose group, Figure 46: Cisplatin + GLP-1 high-dose group

[0428] in conclusion

[0429] Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin can significantly inhibit the growth of tumors in 4T1 breast cancer-bearing mice and has a significant synergistic effect

[0430] Experimental study and data on the killing effect of Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin on LLC lung cancer cells

[0431] Purpose of the experiment:

[0432] To explore the killing effect of Ganoderma lucidum polysaccharide GLP-1 on LLC lung cancer cells and provide experimental basis for its clinical research.

[0433] Experimental Materials

[0434] Test sample

[0435] Ganoderma lucidum polysaccharide GLP-1; batch number: H20220319A, provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0436] Positive control

[0437] Cisplatin, batch number: E2128081, product of Shanghai Aladdin Biochemical Technology Co., Ltd.

[0438] cell lines

[0439] LLC lung cancer cell line was purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0440] Main reagents

[0441] PBS buffer was prepared by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.; 0.25% trypsin was a product of Gibco; MTT was a product of Aladdin; DMSO was a product of Guangdong Guanghua Science and Technology Co., Ltd.; DMEM culture medium was a product of Gibco.

[0442] Main instruments

[0443] 20μL, 200μL, and 1mL pipettes are products of Mettler-Toledo Co., Ltd.; Luna-Ⅱ cell counter is a product of Nanjing Hengqiao Instrument Co., Ltd.; ST-360 microplate reader is a product of Shanghai Kehua Experimental Systems Co., Ltd.

[0444] Experimental methods:

[0445] LLC lung cancer cells were seeded into a 96-well cell culture plate, with 3,000 cells per well. After culturing in a 37°C, 5% carbon dioxide incubator for 24 hours, 2 mg / ml of GLP-1 solution was added to the test sample wells, and 2.5 μg / ml of cisplatin solution was added to the positive wells. After further incubation for 48 hours, the absorbance at 570 nm was measured.

[0446] Test results

[0447] Table 34

[0448] As shown in FIG47 , it shows the distribution of LLC lung cancer cells after 24 hours of culture.

[0449] As shown in FIG48 , it shows the distribution of LLC lung cancer cells after 48 hours of culture.

[0450] As shown in FIG49 , this is the distribution of LLC lung cancer cells in the cisplatin group 24 hours after administration.

[0451] As shown in Figure 50, it shows the distribution of LLC lung cancer cells in the cisplatin group 48 hours after drug administration.

[0452] As shown in Figure 51, it shows the distribution of LLC lung cancer cells in the cisplatin + GLP-1 group 24 hours after drug administration.

[0453] As shown in Figure 52, it shows the distribution of LLC lung cancer cells in the cisplatin + GLP-1 group 48 hours after drug administration.

[0454] in conclusion

[0455] Ganoderma lucidum polysaccharide GLP-1 combined with cisplatin can inhibit the growth of LLC lung cancer cells and enhance the anti-tumor efficacy of cisplatin.

[0456] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Ganoderma lucidum polysaccharide GLP-1, characterized in that: The molecular structure of the Ganoderma lucidum polysaccharide GLP-1 Molecular formula (C 60 H 100 O 50 )n, where n=17-25.

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

3. A method for extracting GLP-1 from Ganoderma lucidum polysaccharide, characterized in that: The method comprises the following steps: S1, removing dust from the ganoderma lucidum, drying it, and then crushing it to make ganoderma lucidum powder; S2, placing the crushed ganoderma lucidum powder in a sealed container, mixing with water and heating, and fully blending the ganoderma lucidum powder and water into a medicinal juice solution under high temperature and high pressure; S3, using membrane concentration technology to separate the medicinal juice solution to obtain a concentrated solution with medicinal ingredients and incompletely fused medicinal residues; S4. The concentrated solution containing the active ingredient is mixed with pure water in a ratio to a water solution of a preset concentration, and subjected to multiple column chromatography separation techniques to obtain Ganoderma lucidum polysaccharide GLP-1 containing the active ingredient.

4. The method for extracting GLP-1 from Ganoderma lucidum polysaccharide according to claim 3, characterized in that: In step S2, the ganoderma lucidum powder and water in the sealed container are mixed and stirred thoroughly, and then heated to 105-200° C. for 2-6 hours. As the heating temperature increases, the pressure inside the sealed container gradually increases to form a high temperature and high pressure environment in the sealed container.

5. The method for extracting GLP-1 from Ganoderma lucidum polysaccharide according to claim 4, characterized in that: In step S2, the mixed liquid of Ganoderma lucidum powder and water in the sealed container is heated to 105-170° C., and the boiling time is continued for 3-6 hours. As the heating temperature increases, the pressure inside the sealed container gradually increases to form a high temperature and high pressure environment of the sealed container.

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

5.

7. The method for extracting GLP-1 from Ganoderma lucidum polysaccharide according to claim 6, characterized in that: In step S3, the aqueous solution containing the extracted medicinal ingredients is concentrated by using membrane concentration technology to remove the Ganoderma lucidum residues therein to obtain a concentrated solution or paste of medicinal ingredients.

8. The method for extracting GLP-1 from Ganoderma lucidum polysaccharide according to claim 7, characterized in that: In the step S1, the ganoderma lucidum is washed with clean water to remove the dust on the surface, and then dried at 105° C. The dried ganoderma lucidum is crushed, and the crushed ganoderma lucidum powder is larger than 60 mesh.

9. The method for extracting GLP-1 from Ganoderma lucidum polysaccharide according to claim 8, characterized in that: In the step S2, the mixed liquid in the sealed container is heated to 105°C, 110°C, 115°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, 170°C, 175°C, 180°C, 185°C, 190°C, 195°C or 200°C, and the boiling time is 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h. As the heating temperature increases, the pressure inside the sealed container gradually increases to form a high temperature and high pressure environment of the sealed container.

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

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