Method for extracting polysaccharide compound from lucid ganoderma, and use of polysaccharide compound

Abstract

The present invention is applicable to the technical field of plant extraction and separation, and provides a method for extracting a polysaccharide compound from lucid ganoderma. The polysaccharide compound is used for treating advanced cancer patients (who have lost the conditions for surgery, have only three to six months left to survive, and are still eligible for chemotherapy), and a lucid ganoderma medicinal component polysaccharide compound is extracted from the polysaccharide compound at high temperature and high pressure. The polysaccharide compound containing the medicinal component has good water solubility, is easily absorbed by human bodies, has an anti-tumor medicinal effect and an effect of preventing the occurrence of tumors in humans, and can eliminate the toxic and side effects of chemotherapeutic drugs on human bodies especially when used in combination with the chemotherapeutic drugs; and there are corresponding experimental data to prove that the polysaccharide compound can control and reduce tumor masses, and reduce and eliminate tumor cells.

Description

Method for extracting polysaccharide compounds from Ganoderma lucidum and uses of the polysaccharide compounds Technical Field

[0001] The present invention belongs to the technical field of plant extraction and separation, and in particular relates to a method for extracting polysaccharide compounds from ganoderma lucidum and uses of the polysaccharide compounds. 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 pharmacological functions from Ganoderma lucidum raw materials and its use.

[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 polysaccharide compounds from Ganoderma lucidum and the polysaccharide compounds, 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, mixing the Ganoderma lucidum powder and water in a sealed container under high temperature and high pressure to fully blend them into a medicinal solution;

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

[0012] S4. The concentrated solution containing the medicinal ingredient is mixed with pure water in a ratio of a preset concentration to obtain an aqueous solution, and the impurities are removed by column chromatography separation technology to obtain a mixed solution polysaccharide compound containing the medicinal ingredient.

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

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

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

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

[0017] A further technical solution of the present invention is: 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 or 170°C, and the boiling time is 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h, and the Ganoderma lucidum powder and water in the sealed container are fully fused into a medicinal juice solution under a high temperature and high pressure environment.

[0018] A further technical solution of the present invention is: the closed container in step S2 is a reactor.

[0019] A further technical solution of the present invention is: the crushing equipment in step S1 is a plant crusher or a plant pulverizer.

[0020] A further technical solution of the present invention is: the cleaning equipment in step S1 is a plant dust removal and cleaning machine, and the drying equipment is a temperature-controlled plant drying machine.

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

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

[0023] FIG1 is a flow chart of a method for extracting polysaccharide compounds from Ganoderma lucidum provided by an embodiment of the present invention.

[0024] FIG2 is a schematic diagram of the anti-tumor model control group of LLC lung cancer-bearing mice treated with Ganoderma lucidum polysaccharide combined with chemotherapy according to an embodiment of the present invention.

[0025] FIG3 is a schematic diagram of the anti-tumor effect of Ganoderma lucidum polysaccharide combined with chemotherapy on the cisplatin group of LLC lung cancer-bearing mice provided by an embodiment of the present invention.

[0026] FIG4 is a schematic diagram of the anti-tumor effect of Ganoderma lucidum polysaccharide combined with chemotherapy on LLC lung cancer-bearing mice in the low-dose cisplatin + GLP group provided by an embodiment of the present invention.

[0027] FIG5 is a schematic diagram of the anti-tumor effect of Ganoderma lucidum polysaccharide combined with chemotherapy on LLC lung cancer-bearing mice in the high-dose cisplatin + GLP group provided by an embodiment of the present invention.

[0028] FIG6 is a schematic diagram of a control group of a tumor model of LLC lung cancer-bearing mice treated with Ganoderma lucidum polysaccharide combined with chemotherapy according to an embodiment of the present invention.

[0029] FIG7 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide combined with chemotherapy on the cisplatin group of LLC lung cancer-bearing mice provided by an embodiment of the present invention.

[0030] FIG8 is a schematic diagram of the cisplatin + GLP low-dose group for the treatment of LLC lung cancer tumor-bearing mice treated with Ganoderma lucidum polysaccharide combined with chemotherapy according to an embodiment of the present invention.

[0031] 9 is a schematic diagram of the cisplatin + GLP high-dose group for the treatment of LLC lung cancer tumor-bearing mice treated with Ganoderma lucidum polysaccharide combined with chemotherapy according to an embodiment of the present invention.

[0032] FIG10 is a schematic diagram of the control group of the anti-tumor model of H22 liver cancer-bearing mice using Ganoderma lucidum polysaccharide combined with chemotherapy provided in an embodiment of the present invention.

[0033] FIG11 is a schematic diagram of the anti-tumor cisplatin group in H22 liver cancer-bearing mice using Ganoderma lucidum polysaccharide combined with chemotherapy provided by an embodiment of the present invention.

[0034] Figure 12 is a schematic diagram of the anti-tumor effect of Ganoderma lucidum polysaccharide combined with chemotherapy on the low-dose cisplatin + GLP group in H22 liver cancer-bearing mice provided by an embodiment of the present invention.

[0035] FIG13 is a schematic diagram of the anti-tumor effect of Ganoderma lucidum polysaccharide combined with chemotherapy on the high-dose cisplatin + GLP group in H22 liver cancer-bearing mice provided by an embodiment of the present invention.

[0036] FIG14 is a schematic diagram of a control group of a tumor model of H22 liver cancer-bearing mice treated with Ganoderma lucidum polysaccharide combined with chemotherapy according to an embodiment of the present invention.

[0037] FIG15 is a schematic diagram of the effect of Ganoderma lucidum polysaccharide combined with chemotherapy on the cisplatin group of H22 liver cancer-bearing mice provided by an embodiment of the present invention.

[0038] 16 is a schematic diagram of the cisplatin + GLP low-dose group for the treatment of H22 liver cancer-bearing mice treated with Ganoderma lucidum polysaccharide combined with chemotherapy according to an embodiment of the present invention.

[0039] 17 is a schematic diagram of the cisplatin + GLP high-dose group for the treatment of H22 liver cancer-bearing mice treated with Ganoderma lucidum polysaccharide combined with chemotherapy according to an embodiment of the present invention. DETAILED DESCRIPTION

[0040] 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.

[0041] In the description of the present invention, it should be understood that the terms "length," "width," "up," "down," "front," "back," "left," "right," "vertical," "horizontal," "top," "bottom," "inside," "outside," and the like, indicating positions or location relationships, are based on the positions or location relationships shown in the accompanying drawings and are intended only to facilitate the description of the present invention and simplify the description. They do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limiting the present invention. Furthermore, in the description of the present invention, "plurality" means two or more, unless otherwise expressly and specifically defined.

[0042] As shown in FIG1 , the flow chart of the method for extracting polysaccharide compounds from Ganoderma lucidum provided by the present invention is described in detail as follows:

[0043] Step S1, washing the harvested Ganoderma lucidum or Ganoderma lucidum that has undergone a preliminary simple treatment with clean water on a washing device to remove dust on the surface thereof, transferring the Ganoderma lucidum to a drying device for high-temperature drying at 105° C., placing the dried Ganoderma lucidum on a plant crusher or a pulverizer to crush it into Ganoderma lucidum powder, sieving the Ganoderma lucidum powder, and transferring the Ganoderma lucidum powder particles larger than 60 mesh to the next process, while the Ganoderma lucidum powder particles smaller than 60 mesh are returned to the crushing mechanism for re-crushing, and the process is repeated multiple times until the crushed Ganoderma lucidum powder particles meet the specified requirements.

[0044] Step S2: mixing the Ganoderma lucidum powder that meets the requirements with pure water and placing the mixture in a sealed container; heating the sealed container containing the mixture; 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 medicinal juice solution; then heating the sealed container to a temperature between 105° C. and 200° C. for a boiling time of 2-6 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 solution is fully fused into a medicinal juice solution.

[0045] Step S3, using membrane concentration technology to separate the medicinal juice solution to obtain a concentrated solution with medicinal ingredients and medicinal residues; the extracted medicinal juice solution with medicinal ingredients is subjected to membrane concentration technology to remove unfused Ganoderma lucidum powder residue to obtain a concentrated solution with medicinal ingredients, wherein the concentrated solution is a paste or a concentrated solution.

[0046] Step S4: The concentrated solution containing the active ingredient is mixed with pure water in a ratio of a preset concentration to obtain a polysaccharide compound of the active ingredient mixture, and the concentrated solution or paste containing the active ingredient is mixed with pure water to obtain an aqueous solution with a concentration ratio of 1:2-1:5, and the prepared aqueous solution containing the active ingredient is subjected to column chromatography separation technology to remove impurities to obtain the polysaccharide compound of the active ingredient mixture.

[0047] The method has simple extraction process, high polysaccharide extraction rate, low production cost and simple operation.

[0048] Another object of the present invention is to provide a use of a polysaccharide compound, which has good water solubility, is easily absorbed by the human body, has anti-tumor efficacy and has a strong effect on preventing the occurrence of tumors in humans. In particular, when used in combination with radiotherapy and chemotherapy drugs, it can eliminate the toxic side effects of radiotherapy and chemotherapy drugs on the human body and has an effect on controlling and reducing tumor masses and reducing and eliminating tumor cells.

[0049] The polysaccharide compound is used in drugs for inhibiting tumor spread or enhancing human immunity.

[0050] The polysaccharide compound includes a mixed solution composed of Ganoderma lucidum polysaccharide GLP-1, Ganoderma lucidum polysaccharide GLP-2 and Ganoderma lucidum polysaccharide GLP-3.

[0051] Experimental study and data on the anti-tumor effect of Ganoderma lucidum polysaccharide combined with chemotherapy on LLC lung cancer-bearing mice

[0052] Purpose of the experiment

[0053] C57 mice were inoculated with LLC lung cancer tumor homogenate in the right axilla to prepare tumor-bearing mice. The anti-tumor effect of Ganoderma lucidum polysaccharide on lung cancer tumor-bearing mice was studied to provide experimental basis for its clinical research.

[0054] Experimental Materials

[0055] Test sample

[0056] Ganoderma lucidum polysaccharide GLP was provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0057] Positive control

[0058] Cisplatin, batch number: L2107580, was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0059] experimental animals

[0060] Seventy 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 (Guangdong) 2022-0002, and the laboratory animal quality certificate number is 44007200118349.

[0061] Main reagents

[0062] PBS buffer was prepared by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.; fetal bovine serum was a product of Zhejiang Tianhang Biotechnology Co., Ltd.; DMEM culture medium was a product of Gibco; and 0.25% trypsin was a product of Gibco.

[0063] Main instruments

[0064] Vernier caliper, product of Shanghai Tool Factory Co., Ltd.; I-2000 balance, product of Dongguan Nancheng Changxie Electronic Products Factory; ophthalmic scissors and tweezers, product of Shanghai Jinzhong Medical Instrument Co., Ltd.; CCL-170B-8 carbon dioxide incubator, product of Singapore ESCO; Luna-Ⅱ cell counter, product of Nanjing Hengqiao Instrument Co., Ltd.; JRA-35S handheld homogenizer, product of Wuxi Jerian Instrument Equipment Co., Ltd.

[0065] Experimental methods

[0066] Normally grown LLC cells were inoculated subcutaneously on the left shoulder of 5 healthy male C57 mice. 3 When the tumor mass was removed and made into a homogenate suspension, it was injected into the axillary subcutaneous tissue of 50 healthy male C57 mice to establish a solid tumor model. 3 At approximately 60 days, mice were randomly divided into groups based on tumor volume and given the corresponding drug or vehicle by gavage or intraperitoneal injection for 17 consecutive days. Tumor length and volume were measured every 3 days, and mice were weighed every 3 days. At the end of the experiment, tumors, spleens, and thymuses were removed and weighed, and the tumor index, spleen index, and thymus index were calculated.

[0067] Dosage design

[0068] GLP was designed based on previous experimental results. The low dose was 50 mg / kg and the high dose was 150 mg / kg. The corresponding drugs were administered as shown in Table 1.

[0069] The basis for designing the cisplatin dosage is as follows: Based on the clinical dosage of cisplatin not exceeding 100 mg / m2 per person per day and the tolerance of mice to cisplatin, a dose of 4 mg / kg was selected as the dosage.

[0070] Table 1 Trial grouping and dosage design

[0071] Detection indicators

[0072] Efficacy indicators

[0073] Relative tumor inhibition rate

[0074] 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.

[0075] Tumor growth inhibition rate

[0076] 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.

[0077] Spleen and thymus organ coefficients: After the final dose, the spleen, thymus, and tumor were weighed and the organ coefficients were calculated. Tumor index (%) = tumor weight / body weight × 100%. Immune organ index (mg / g) = organ weight / body weight × 1000.

[0078] Data processing and statistical analysis

[0079] 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.

[0080] Experimental results

[0081] Animal deaths

[0082] As shown in Table 2, the mortality rate of mice in the model control group of this experiment was 25%, the mortality rate of mice in the cisplatin + GLP high-dose group was 12.5%, and the mortality rate of mice in the other groups was 0.

[0083] Table 2 Statistics of the number of surviving animals and mortality rates in each group

[0084] Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on body weight in LLC lung cancer-bearing mice

[0085] As shown in Table 3, compared with the normal group, the body weight of mice in the model control group increased significantly on D3 and D12-D17, and the body weight of mice in the cisplatin group and the cisplatin combined with low-dose and high-dose GLP groups decreased significantly on D6-D17.

[0086] Compared with the model control group, the body weight of mice in the cisplatin group and the cisplatin combined with low-dose and high-dose GLP groups was significantly reduced from D3 to D17.

[0087] Compared with the cisplatin group, the body weight of mice in the low-dose cisplatin + GLP group was significantly reduced on D15, and the body weight of mice in the high-dose cisplatin + GLP group was significantly reduced on D12.

[0088] Table 3 Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on body weight of LLC lung cancer-bearing mice Note: Compared with the model control group, + P<0.05; compared with the cisplatin group, # P<0.05; compared with the normal group, * P<0.05.

[0089] Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on tumor volume in LLC lung cancer-bearing mice

[0090] As shown in Table 4, compared with the model control group, the tumor volume of mice in the cisplatin group and the low-dose cisplatin + GLP group was significantly reduced from D3 to D17, and the tumor volume of mice in the high-dose cisplatin + GLP group was significantly reduced from D6 to D17. Compared with the cisplatin group, the tumor volume of mice in the low-dose cisplatin + GLP group was significantly reduced from D12 to D17.

[0091] Table 4 Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on tumor volume in LLC lung cancer-bearing mice Note: Compared with the model control group, + P<0.05; compared with the cisplatin group, # P<0.05.

[0092] Effect of Ganoderma lucidum polysaccharide combined with chemotherapy on relative tumor inhibition rate in LLC lung cancer-bearing mice

[0093] As shown in Table 5, compared with the model control group, the relative tumor inhibition rates of the cisplatin group were above 40% on D6 to D17, which were 49.5%, 65.7%, 71.9%, 77.6% and 80.8% respectively; the relative tumor inhibition rates of the cisplatin + GLP low-dose group were above 40% on D3 to D17, which were 43.9%, 53.6%, 74.2%, 81.8%, 85.5% and 87.4% respectively; the relative tumor inhibition rates of the cisplatin + GLP high-dose group were above 40% on D9 to D17, which were 53.2%, 73.0%, 80.6% and 84.1% respectively.

[0094] As shown in Table 6, compared with the cisplatin group, the relative tumor inhibition rates of the cisplatin + GLP low-dose group were 35.4%, 35.1%, and 34.5% on D12 to D17 (P<0.05).

[0095] Table 5 Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on the relative tumor inhibition rate of LLC lung cancer-bearing mice (compared with the model control group) Note: Compared with the model control group, + P<0.05.

[0096] Table 6 Effect of Ganoderma lucidum polysaccharide combined with chemotherapy on the relative tumor inhibition rate of LLC lung cancer-bearing mice (compared with the cisplatin group) Note: Compared with cisplatin group, # P<0.05.

[0097] Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on organ index and tumor growth inhibition rate in LLC lung cancer-bearing mice

[0098] As shown in Table 7, compared with the model control group, the tumor index, spleen index, and thymus index in the cisplatin group, the low-dose cisplatin + GLP group, and the high-dose cisplatin + GLP group were significantly reduced. Compared with the cisplatin group, the thymus index in the low-dose cisplatin + GLP group was significantly increased. Compared with the normal group, the spleen index in the model control group and the cisplatin group was significantly increased, while the thymus index in the model control group, the cisplatin group, the low-dose cisplatin + GLP group, and the high-dose cisplatin + GLP group was significantly decreased.

[0099] Compared with the model control group, the tumor growth inhibition rates in the cisplatin group were 75.5%, 83.5% in the low-dose cisplatin + GLP group, and 82.3% in the high-dose cisplatin + GLP group. Compared with the cisplatin group, the tumor growth inhibition rates in the low-dose cisplatin + GLP group and the high-dose cisplatin + GLP group were 32.6% and 27.8%, respectively.

[0100] Table 7 Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on organ index and tumor growth inhibition rate in LLC lung cancer-bearing mice Note: Compared with the model control group, + P<0.05; compared with the cisplatin group, # P<0.05; compared with the normal group, * P<0.05.

[0101] Figures 2, 3, 4, and 5 are pictures of tumor-bearing mice, and the corresponding groups are: Figure 2: Model control group, Figure 3: Cisplatin group, Figure 4: Cisplatin + GLP low-dose group, Figure 5: Cisplatin + GLP high-dose group

[0102] Figures 6, 7, 8, and 9 show tumor images of tumor-bearing mice, and the corresponding groups are: Figure 6: Model control group, Figure 7: Cisplatin group, Figure 8: Cisplatin + GLP low-dose group, Figure 9: Cisplatin + GLP high-dose group

[0103] in conclusion

[0104] GLP combined with cisplatin can significantly inhibit the growth of tumors in LLC lung cancer-bearing mice and has a significant synergistic effect.

[0105] Experimental study and data on the anti-tumor effect of Ganoderma lucidum polysaccharide combined with chemotherapy on H22 liver cancer-bearing mice

[0106] Purpose of the experiment

[0107] The H22 liver cancer homogenate was inoculated into the right axilla of C57 mice to prepare tumor-bearing mice. The anti-tumor effect of Ganoderma lucidum polysaccharide on liver cancer-bearing mice was studied to provide experimental basis for its clinical research.

[0108] Experimental Materials

[0109] Test sample

[0110] Ganoderma lucidum polysaccharide GLP was provided by Shenzhen Aolimei Oncology Medical Technology Co., Ltd.

[0111] Positive control

[0112] Cisplatin, batch number: E2128081, product of Shanghai Aladdin Biochemical Technology Co., Ltd.

[0113] experimental animals

[0114] Seventy 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 44007200110321.

[0115] Main reagents

[0116] 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.

[0117] Main instruments

[0118] Vernier caliper, product of Shanghai Tool Factory Co., Ltd.; I-2000 balance, product of Dongguan Nancheng Changxie Electronic Products Factory; ophthalmic scissors and tweezers, product of Shanghai Jinzhong Medical Instrument Co., Ltd.; CCL-170B-8 carbon dioxide incubator, product of Singapore ESCO; Luna-Ⅱ cell counter, product of Nanjing Hengqiao Instrument Co., Ltd.; JRA-35S handheld homogenizer, product of Wuxi Jerian Instrument Equipment Co., Ltd.

[0119] Experimental methods

[0120] The H22 liver cancer solid tumor homogenate suspension was injected subcutaneously into the axilla of 55 healthy male C57 mice to establish a solid tumor model. 3 At approximately 60 days, mice were randomly divided into groups based on tumor volume and given the corresponding drug or vehicle by gavage or intraperitoneal injection for 18 consecutive days. Tumor diameters were measured every 3 days to calculate tumor volume, and mice were weighed every 3 days. At the end of the experiment, tumors, spleens, and thymuses were removed and weighed, and the tumor index, spleen index, and thymus index were calculated.

[0121] Dosage design

[0122] GLP was designed based on previous experimental results. The low dose was 50 mg / kg and the high dose was 150 mg / kg. The corresponding drugs were administered as shown in Table 8.

[0123] The basis for designing the cisplatin dosage is as follows: Based on the clinical dosage of cisplatin not exceeding 100 mg / m2 per person per day and the tolerance of mice to cisplatin, a dose of 3 mg / kg was selected as the dosage.

[0124] Table 8 Trial grouping and dosage design

[0125] Detection indicators

[0126] Efficacy indicators

[0127] Relative tumor inhibition rate

[0128] 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.

[0129] Tumor growth inhibition rate

[0130] 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.

[0131] Spleen and thymus organ coefficients: After the final dose, the spleen, thymus, and tumor were weighed and the organ coefficients were calculated. Tumor index (%) = tumor weight / body weight × 100%. Immune organ index (mg / g) = organ weight / body weight × 1000.

[0132] Data processing and statistical analysis

[0133] SPSS 17.0 was used for statistical analysis. The level of statistical significance was set at P ≤ 0.05. Measurement data were expressed as mean ± standard deviation (x ± s). 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.

[0134] Experimental results

[0135] Animal deaths

[0136] As shown in Table 9, the mortality rate of mice in the cisplatin group was 12.5%, and the mortality rate of mice in the other groups was 0.

[0137] Table 9 Statistics of the number of surviving animals and mortality rates in each group

[0138] Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on body weight in H22 liver cancer-bearing mice

[0139] As shown in Table 10, compared with the model control group, the body weight of mice in the cisplatin group was significantly reduced from D12 to D18, and the body weight of mice in the low-dose and high-dose cisplatin + GLP groups was significantly reduced from D6 to D18. Compared with the cisplatin group, the body weight of mice in the low-dose and high-dose cisplatin + GLP groups was significantly reduced from D9 to D18. Compared with the normal group, the body weight of mice in the cisplatin group was significantly reduced from D9 to D18, and the low-dose and high-dose cisplatin + GLP groups was significantly reduced from D6 to D18.

[0140] Table 10 Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on body weight of H22 liver cancer-bearing mice Note: Compared with the model control group, + P<0.05; compared with the cisplatin group, # P<0.05; compared with the normal group, * P<0.05.

[0141] Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on tumor volume in H22 liver cancer-bearing mice

[0142] As shown in Table 11, compared with the model control group, the tumor volumes of mice in the cisplatin group, the cisplatin + GLP low-dose group, and the cisplatin + GLP high-dose group were significantly reduced from D6 to D18.

[0143] Compared with the cisplatin group, there was no significant difference in the tumor volume of mice in the low-dose and high-dose cisplatin + GLP groups.

[0144] Table 11 Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on tumor volume in H22 liver cancer-bearing mice Note: Compared with the model control group, + P<0.05; compared with the cisplatin group, # P<0.05.

[0145] Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on relative tumor inhibition rate in H22 liver cancer-bearing mice

[0146] As shown in Table 12, compared with the model control group, the relative tumor inhibition rate of the cisplatin group was above 40% on D6 to D18, which were 49.9%, 40.8%, 50.2%, 66.3% and 73.0% respectively; the relative tumor inhibition rate of the cisplatin + GLP low-dose group was above 50% on D6 to D18, which were 55.4%, 53.4%, 59.9%, 79.5% and 78.9% respectively; the relative tumor inhibition rate of the cisplatin + GLP high-dose group was basically above 59% on D6 to D18, which were 59.9%, 61.3%, 67.0%, 78.1% and 80.9% respectively.

[0147] As shown in Table 13, compared with the cisplatin group, the relative tumor inhibition rate of the low-dose cisplatin + GLP group was above 20% on D9, D15, and D18, and the relative tumor inhibition rate of the high-dose cisplatin + GLP group was above 20% on D6 to D18.

[0148] Table 12 Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on the relative tumor inhibition rate of H22 liver cancer-bearing mice (compared with the model control group) Note: Compared with the model control group, + P<0.05.

[0149] Table 13 Effect of Ganoderma lucidum polysaccharide combined with chemotherapy on the relative tumor inhibition rate of H22 liver cancer-bearing mice (compared with the cisplatin group) Note: Compared with cisplatin group, # P<0.05.

[0150] Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on organ index and tumor growth inhibition rate in H22 liver cancer-bearing mice

[0151] As shown in Table 14, compared with the model group, the tumor index, spleen index, and thymus index were significantly decreased in the cisplatin group, the low-dose cisplatin + GLP group, and the high-dose cisplatin + GLP group. Compared with the cisplatin group, the thymus index in the high-dose cisplatin + GLP group was significantly decreased. Compared with the normal group, the spleen index in the model control group was significantly increased, while the thymus index in the cisplatin group, the low-dose cisplatin + GLP group, and the high-dose cisplatin + GLP group was significantly decreased.

[0152] Compared with the model control group, the tumor growth inhibition rates in the cisplatin group were 80.0%, 82.7% in the low-dose cisplatin + GLP group, and 82.6% in the high-dose cisplatin + GLP group. Compared with the cisplatin group, the tumor growth inhibition rates in the low-dose cisplatin + GLP group and the high-dose cisplatin + GLP group were 13.5% and 12.7%, respectively.

[0153] Table 14 Effects of Ganoderma lucidum polysaccharide combined with chemotherapy on organ index and tumor growth inhibition rate in H22 liver cancer-bearing mice Note: Compared with the model control group, + P<0.05; compared with the cisplatin group, # P<0.05; compared with the normal group, * P<0.05.

[0154] Figures 10, 11, 12, and 13 are pictures of tumor-bearing mice, and the corresponding groups are: Figure 10: Model control group, Figure 11: Cisplatin group, Figure 12: Cisplatin + GLP low-dose group, Figure 13: Cisplatin + GLP high-dose group

[0155] Figures 14, 15, 16, and 17 show tumor images of tumor-bearing mice, and the corresponding groups are: Figure 14: Model control group, Figure 15: Cisplatin group, Figure 16: Cisplatin + GLP low-dose group, Figure 17: Cisplatin + GLP high-dose group

[0156] in conclusion

[0157] GLP combined with cisplatin can significantly inhibit the growth of tumors in H22 liver cancer-bearing mice and has a synergistic effect.

[0158] 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 method for extracting polysaccharide compounds from Ganoderma lucidum, 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, mixing the ganoderma lucidum powder with water, placing them in a sealed container under high temperature and high pressure to fully fuse them into a medicinal juice solution; S3, using membrane concentration technology to separate the medicinal juice solution to obtain a concentrated solution with medicinal components and medicinal residues; S4. The concentrated solution containing the active ingredient is mixed with pure water in a ratio to a preset concentration of aqueous solution, and the impurities are removed by column chromatography separation technology to obtain a mixed solution polysaccharide compound containing the active active ingredient.

2. The method for extracting polysaccharide compounds from Ganoderma lucidum according to claim 1, characterized in that: In step S2, the ganoderma powder and water are mixed and fully heated to 105-200° C., and the boiling time lasts for 2-6 hours. The ganoderma powder and water in the sealed container are fully fused into a medicinal juice solution under high temperature and high pressure.

3. The method for extracting polysaccharide compounds from Ganoderma lucidum according to claim 1, characterized in that: In step S2, the ganoderma powder and water are mixed and fully heated to 105-170° C., and the boiling time lasts for 3-6 hours. The ganoderma powder and water in the sealed container are fully fused into a medicinal juice solution under high temperature and high pressure.

4. The method for extracting polysaccharide compounds from Ganoderma lucidum according to claim 2 or 3, characterized in that: In the step S4, the concentrated solution containing the medicinal ingredients is mixed with pure water to form an aqueous solution with a concentration ratio of 1:2 to 1:

5.

5. The method for extracting polysaccharide compounds from Ganoderma lucidum according to claim 4, characterized in that: In step S1, the ganoderma lucidum is washed with clean water to remove the dust on the surface, and then dried at a high temperature of 105° C. by a drying device. The dried ganoderma lucidum is placed in a crushing device for crushing to obtain ganoderma lucidum powder, wherein the ganoderma lucidum powder is larger than 60 meshes.

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

7. The method for extracting polysaccharide compounds from Ganoderma lucidum according to claim 6, characterized in that: The sealed container in step S2 is a reactor.

8. The method for extracting polysaccharide compounds from Ganoderma lucidum according to claim 7, characterized in that: The crushing equipment in step S1 is a plant crusher or a plant pulverizer.

9. The method for extracting polysaccharide compounds from Ganoderma lucidum according to claim 9, characterized in that: The cleaning equipment in step S1 is a plant dust removal cleaning machine, and the drying equipment is a temperature-controlled plant drying machine.

10. Use of the method of claims 1 to 9 for extracting polysaccharide compounds from Ganoderma lucidum, characterized in that: The polysaccharide compound 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 radiotherapy and chemotherapy drugs, it can eliminate the toxic side effects of radiotherapy and chemotherapy drugs on the human body and has an effect on controlling and reducing tumor masses and reducing and eliminating tumor cells.

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