Ganoderma lucidum polysaccharide and its preparation method and application

Abstract

The present invention discloses a ganoderma polysaccharide and a preparation method and application thereof. The present invention uses a combination of water extraction and alcohol precipitation method and alkali extraction and alcohol precipitation method to perform preliminary separation of ganoderma polysaccharide to obtain a variety of ganoderma crude polysaccharides, and further uses ion exchange chromatography and molecular sieve gel column chromatography method to purify the ganoderma crude polysaccharide, and prepares a new ganoderma fine polysaccharide for the first time, including at least one of GSP4‑2, GSP‑4‑1‑2 and GSP‑4‑1‑3, and systematically analyzes and confirms its physicochemical properties, molecular weight, monosaccharide composition, etc., and successfully obtains the characteristic structure of the fine polysaccharide. The present invention also finds that ganoderma crude polysaccharide and ganoderma fine polysaccharide can inhibit the proliferation, invasion and metastasis of triple-negative breast cancer cells, and inhibit angiogenesis, and can be used to prepare drugs, health products or functional foods for preventing and / or treating triple-negative breast cancer or improving the quality of life of patients.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a Ganoderma lucidum polysaccharide and a preparation method and application thereof. Background Art

[0002] Cancer is a major public health issue and has become one of the most common causes of death in China. Breast cancer is the most common malignant tumor in women and has become the world's leading cancer. In 2020, there were 2.3 million new cases of breast cancer, accounting for 11.7% of new cancers. Triple-negative breast cancer (TNBC) refers to breast cancer in which estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) are all negative, accounting for about 15-20% of the breast cancer population. TNBC is a molecular subtype with the worst prognosis among breast cancer patients. It has the characteristics of strong invasiveness, easy metastasis, high recurrence rate, and lack of targeted therapy receptors. According to the European Institute of Oncology, 20-30% of early breast cancer patients will develop metastatic disease, and 5-10% of patients have metastasis at the time of diagnosis. The 5-year survival rate of women with metastatic breast cancer ranges from 18 to 36%, while the 5-year survival rate of patients with non-metastatic breast cancer exceeds 90%. Therefore, TNBC has become a focus issue that needs to be urgently addressed in the clinic. Chemotherapy is the main treatment for TNBC. Paclitaxel / anthracycline drugs are the first choice for chemotherapy. Platinum drugs combined with paclitaxel are effective adjuvant chemotherapy options for patients with operable TNBC. Although the above-mentioned chemotherapy drugs prolong the survival period of TNBC, they have serious toxic side effects. In addition, TNBC is prone to metastasis. TNBC cells can obtain sufficient oxygen and nutrient supply by promoting angiogenesis, making it easy for tumor cells in the primary lesion to migrate to other tissues, leading to secondary tumor proliferation, which increases the difficulty of treatment. Epithelial-mesenchymal transition (EMT) refers to the process of mucosal epithelial cells transforming from a well-polarized cell state to a non-polarized cell with a tendency to embed in the extracellular matrix (ECM), which plays a vital role in the development of the body and the differentiation of various tissues and organs. The EMT program is coordinated by a group of multidirectional transcription factors, including Slug, Snail, Twist, ZEB1 and ZEB2, which mobilize cells into the mesenchymal state by inhibiting the expression of epithelial markers and inducing the expression of other markers related to the mesenchymal state. EMT plays an important role in tumor invasion, metastasis and recurrence. Tumor cells acquire morphological and functional changes through EMT, which enables tumor cells to acquire the ability of mesenchymal-like cells, drive them to invade and metastasize, and eventually cause metastatic lesions in the host. The invasion and metastasis of TNBC are closely related to EMT. At present, there are very few types of drugs on the market for invasive tumors, with limited clinical efficacy and certain usage restrictions and corresponding toxic side effects. Traditional Chinese medicine is a natural treasure of my country, with rich resources, a wide variety of varieties, and a long history of development. It is relatively complete in theory and practical experience and has huge development potential.In the research and development of anti-tumor drugs, anti-tumor drugs derived from active ingredients in traditional Chinese medicine, such as paclitaxel, camptothecin, vinblastine, podophyllotoxin, lentinan and Sophora japonica polysaccharide, play an important role in the treatment of tumors. Therefore, the prospect of finding and discovering anti-tumor drugs from traditional Chinese medicine is very broad. As one of the effective ingredients of traditional Chinese medicine, traditional Chinese medicine polysaccharides have multiple effects such as immunomodulation and anti-tumor. Obtaining high-efficiency, low-toxic polysaccharides with anti-tumor activity from traditional Chinese medicine has attracted increasing attention.

[0003] Ganoderma lucidum belongs to the Polyporaceae family of the Basidiomycetes. It is a traditional precious Chinese medicine with a long history of medicinal use. It is known as the "Four Great Immortal Grasses" together with ginseng, Polygonum multiflorum, and Cordyceps sinensis. According to the "Chinese Pharmacopoeia" (2020 edition), the dried fruiting bodies of Ganoderma lucidum (Leyss. ex Fr.) Karst. and Ganoderma sinense Zhao, Xu et Zhang are the legal sources of the traditional Chinese medicine Ganoderma lucidum. Ganoderma lucidum is sweet and flat in nature. It is regulated by the heart, lung, liver, and kidney meridians. It has the effects of replenishing qi and calming the mind, relieving cough and relieving asthma. Modern pharmacological studies have shown that Ganoderma lucidum has a wide range of biological activities and can be used to prevent and treat a variety of diseases. Ganoderma lucidum has a rich variety of chemical components, mainly including polysaccharides, triterpenes, nucleotides, steroids, polypeptides, fatty acids, and amino acids. Ganoderma polysaccharides, as an important bioactive component in Ganoderma lucidum, have biological activities such as immunomodulation, anti-tumor, liver protection, hypoglycemic, neuroprotection, antioxidant, and cardiovascular system protection. However, there are few reports on the selective inhibition of triple-negative breast cancer cell proliferation, invasion and metastasis by Ganoderma lucidum polysaccharides. Summary of the invention

[0004] The purpose of the first aspect of the present invention is to provide a Ganoderma lucidum polysaccharide, including at least one of GSP4-2, GSP-4-1-2 and GSP-4-1-3.

[0005] The second aspect of the present invention aims to provide a method for preparing crude polysaccharide of Ganoderma lucidum.

[0006] The third aspect of the present invention is to provide a crude polysaccharide of Ganoderma lucidum.

[0007] The fourth aspect of the present invention aims to provide a method for preparing the above-mentioned Ganoderma lucidum polysaccharide.

[0008] The fifth aspect of the present invention aims to provide the application of the above-mentioned Ganoderma lucidum polysaccharide and Ganoderma lucidum crude polysaccharide.

[0009] The sixth aspect of the present invention aims to provide a product.

[0010] The technical solution adopted by the present invention is:

[0011] In a first aspect of the present invention, there is provided a ganoderma lucidum polysaccharide, comprising at least one of GSP4-2, GSP-4-1-2 and GSP-4-1-3, wherein the structural formula of the ganoderma lucidum polysaccharide GSP4-2 is as shown in formula (I):

[0012]

[0013] Among them, the range of a, b, c, d, and e is 1 to 100.

[0014] Preferably, the molecular weight of the Ganoderma lucidum polysaccharide GSP4-2 is in the range of 10k to 200k Da.

[0015] Preferably, the Ganoderma lucidum polysaccharide GSP4-2 includes mannose, glucuronic acid, glucose, galactose, and fucose.

[0016] Preferably, the molar ratio of mannose, glucuronic acid, glucose, galactose, and fucose is (7-9):(2.5-3.5):(19-21):(3.5-5):1.

[0017] Preferably, the molar ratio of mannose, glucuronic acid, glucose, galactose, and fucose is 8.0:3.2:19.7:4.3:1.0.

[0018] The structural formula of the Ganoderma lucidum polysaccharide GSP4-1-2 is shown in formula (II):

[0019]

[0020] Among them, the range of a, b, c, d, and e is 1 to 100.

[0021] Preferably, the molecular weight of the Ganoderma lucidum polysaccharide GSP4-1-2 is in the range of 7k to 100k Da.

[0022] Preferably, the Ganoderma lucidum polysaccharide GSP4-1-2 includes mannose, glucose, galactose, xylose and fucose.

[0023] Preferably, the molar ratio of mannose, glucose, galactose, xylose and fucose is (20-22):(15-17):(48-55):(3-5):(7-9).

[0024] Preferably, the molar ratio of mannose, glucose, galactose, xylose and fucose is 21.1:16.3:51.2:4.1:8.2.

[0025] The structural formula of the Ganoderma lucidum polysaccharide GSP4-1-3 is shown in formula (III):

[0026]

[0027] Among them, the range of a, b, c, d, and e is 1 to 100.

[0028] Preferably, the molecular weight of the Ganoderma lucidum polysaccharide GSP4-1-3 is in the range of 10k to 200k Da.

[0029] Preferably, the Ganoderma lucidum polysaccharide GSP4-1-3 includes mannose, glucose, galactose and fucose.

[0030] Preferably, the molar ratio of mannose, glucose, galactose and fucose is (4.5-6):(3-5):(48-56):(4-7).

[0031] Preferably, the molar ratio of mannose, glucose, galactose and fucose is 5.4:4.1:42.8:5.4.

[0032] The second aspect of the present invention provides a method for preparing Ganoderma lucidum crude polysaccharide, comprising method (I) and / or method (II);

[0033] Method (I) comprises the following steps:

[0034] S11: extracting Ganoderma lucidum with water and collecting the extract;

[0035] S12: subjecting the extract obtained in step S11 to graded alcohol precipitation to obtain crude polysaccharide GS1, crude polysaccharide GS2, and crude polysaccharide GS3;

[0036] S13: removing protein from the crude polysaccharide GS1, crude polysaccharide GS2 and crude polysaccharide GS3 described in step S12 to obtain crude polysaccharide GS1, crude polysaccharide GS2 and crude polysaccharide GS3 of Ganoderma lucidum;

[0037] Method (II) comprises the following steps:

[0038] S21: extracting Ganoderma lucidum with water and collecting the obtained residue;

[0039] S22: extracting the residue obtained in step S21 with alkali to obtain crude polysaccharide GS4;

[0040] S23: removing the protein in the crude polysaccharide GS4 described in step S22 to obtain the crude polysaccharide GS4 of Ganoderma lucidum.

[0041] Preferably, in step S11 and step S21, the Ganoderma lucidum is soaked in water in advance.

[0042] Preferably, the Ganoderma lucidum used for soaking is dried Ganoderma lucidum.

[0043] Preferably, the dried Ganoderma lucidum is divided into small pieces of 2 to 5 cm.

[0044] Preferably, the soaking time is 6 to 12 hours.

[0045] Preferably, the specific operation of water extraction in step S12 and step S21 includes: mixing the soaked Ganoderma lucidum with water, and extracting at 60-100° C. for 1-10 hours.

[0046] Preferably, the volume ratio of Ganoderma lucidum to water is 1:(5-15).

[0047] Preferably, the specific operation of the graded alcohol precipitation in step S12 includes: concentrating the extract obtained in step S1, adding ethanol to a volume concentration of A%, standing to obtain a supernatant and a precipitate, and collecting the precipitate to obtain crude polysaccharide GS1; concentrating the supernatant, adding ethanol to a volume concentration of B%, standing to obtain a supernatant and a precipitate, and collecting the precipitate to obtain crude polysaccharide GS2; and concentrating the supernatant again, adding ethanol to a volume concentration of C%, standing to collect the precipitate, and obtaining crude polysaccharide GS3.

[0048] Preferably, 10≤A<B<C<100.

[0049] Preferably, 10≤A<60, 60≤B<80, 80≤C<100.

[0050] Preferably, the standing time is 10 to 28 hours.

[0051] Preferably, the specific operation of step S22 includes: soaking the residue described in step S21 in 0.1-1M NaOH solution for alkali extraction, collecting the alkali extract, and adjusting the alkali extract to a pH of 6.0-8.0, obtaining a supernatant after solid-liquid separation, and precipitating the supernatant with alcohol to obtain crude polysaccharide GS4.

[0052] Preferably, the volume ratio of the residue to the sodium hydroxide solution is 1:(5-20).

[0053] Preferably, the conditions for the alkaline extraction include: standing at room temperature for 1 to 4 hours.

[0054] Preferably, the solid-liquid separation method includes centrifugation.

[0055] Preferably, the centrifugation condition is 1500-2000×g, 8-12 min.

[0056] Preferably, the alcohol precipitation method is to mix the supernatant with ethanol and let the mixture stand.

[0057] Preferably, the volume concentration of ethanol in the precipitated alcohol is 30-100%.

[0058] Preferably, the standing time in the alcohol precipitation is 10 to 28 hours.

[0059] Preferably, the protein removal operation in step S13 and step S23 includes: removing protein using the Sevag method.

[0060] Preferably, the protein is removed and then dialyzed and dried.

[0061] Preferably, the molecular weight cut-off of the dialysis is 100 to 1200 Da;

[0062] Preferably, method (II) further comprises: further purifying the crude polysaccharide GS4 from Ganoderma lucidum to obtain GSP4-2, GSP-4-1-2 and GSP-4-1-3 as described in the first aspect of the present invention.

[0063] Preferably, the purification includes purification by ion exchange column chromatography and molecular sieve gel column chromatography;

[0064] Preferably, the ion exchange column chromatography comprises ion exchange cellulose or ion exchange gel.

[0065] Preferably, the ion exchange column chromatography uses a 0-2 M NaCl solution for gradient elution.

[0066] Preferably, the ion exchange column chromatography uses 0, 0.05, 0.15, 0.2 M NaCl solutions for gradient elution.

[0067] Preferably, after the ion exchange chromatography, the sugar portion is collected according to the elution curve, then concentrated and freeze-dried, and then dissolved in water and centrifuged to obtain a supernatant.

[0068] Preferably, the supernatant obtained after ion exchange chromatography is subjected to molecular sieve gel chromatography.

[0069] Preferably, the molecular sieve gel column chromatography includes Sephadex G or Sephacryl S series molecular sieve column chromatography.

[0070] Preferably, the molecular sieve gel column chromatography is eluted with water.

[0071] Preferably, after the molecular sieve gel chromatography, the sugar portion is collected according to the elution curve, and then concentrated and freeze-dried to obtain Ganoderma lucidum polysaccharide.

[0072] Preferably, the concentration is to 1 / 9 to 1 / 13 of the original volume.

[0073] The third aspect of the present invention provides a crude polysaccharide of Ganoderma lucidum, characterized in that the crude polysaccharide of Ganoderma lucidum is prepared by the method described in the second aspect of the present invention.

[0074] The fourth aspect of the present invention provides the use of the Ganoderma lucidum polysaccharide described in the first aspect of the present invention or the Ganoderma lucidum crude polysaccharide described in the third aspect of the present invention in preparing products.

[0075] Preferably, the products include health products, food, daily chemicals and medicines.

[0076] Preferably, the drug is used to inhibit the proliferation, invasion, and metastasis of triple-negative breast cancer cells, prevent and / or treat triple-negative breast cancer, improve the quality of life of patients, and inhibit angiogenesis.

[0077] The fifth aspect of the present invention provides a product, wherein the product comprises the Ganoderma lucidum polysaccharide described in the first aspect of the present invention or the crude polysaccharide described in the third aspect of the present invention.

[0078] Preferably, the products include health products, food, daily chemicals and medicines.

[0079] Preferably, the medicine and / or food and / or health product is in the form of oral solution, oral suspension, capsule, tablet, powder or granule.

[0080] Preferably, the daily chemical product is in the form of an aqueous solution, emulsion, ointment, cream, foam, lotion, roll-on, gel or spray.

[0081] Preferably, the product also includes excipients acceptable to medicines, health products or daily chemicals.

[0082] The beneficial effects of the present invention are:

[0083] The invention combines a water extraction and alcohol precipitation method with an alkali extraction and alcohol precipitation method, performs graded alcohol precipitation from low to high ethanol concentration, and performs preliminary separation of Ganoderma lucidum polysaccharide. At the same time, high-concentration ethanol can separate polysaccharides with high polarity and good water solubility from polysaccharides with low polarity and poor water solubility, so that the extracted polysaccharide composition contains more types of polysaccharide components, and a variety of Ganoderma lucidum crude polysaccharides are obtained. The operation is simple and convenient, and large-scale production can be carried out.

[0084] The present invention further utilizes ion exchange chromatography and molecular sieve gel chromatography methods to purify crude Ganoderma lucidum polysaccharide, and prepares for the first time a new Ganoderma lucidum polysaccharide, including at least one of GSP4-2, GSP-4-1-2 and GSP-4-1-3, and systematically analyzes and confirms its physicochemical properties, molecular weight, monosaccharide composition, etc., and successfully obtains the characteristic structure of the polysaccharide.

[0085] The present invention also found that Ganoderma lucidum crude polysaccharide and Ganoderma lucidum essence polysaccharide can inhibit the proliferation, invasion and metastasis of triple-negative breast cancer cells and inhibit angiogenesis, and can be used to prepare drugs, health products or functional foods for preventing and / or treating triple-negative breast cancer or improving the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 : Infrared spectrum of GSP4-2.

[0087] Figure 2 :GSP4-2 1 H NMR spectrum.

[0088] Figure 3 :GSP4-2 13 C NMR spectrum.

[0089] Figure 4 :GSP4-2 1 H- 1 H COSY spectrum.

[0090] Figure 5 : HSQC spectrum of GSP4-2.

[0091] Figure 6 : HMBC spectrum of GSP4-2.

[0092] Figure 7 :NMR spectrum of GSP4-1-2. Figures (A), (B), (C), (D), and (E) respectively represent the 1 H NMR spectrum, 13 C NMR spectrum, HSQC spectrum, HMBC spectrum, 1 H- 1 H COSY spectrum.

[0093] Figure 8 :NMR spectrum of GSP4-1-3. Figures (A), (B), (C), (D), and (E) respectively represent the 1 H NMR spectrum, 13 C NMR spectrum, HSQC spectrum, HMBC spectrum, 1 H- 1 H COSY spectrum.

[0094] Fig. 9 :Effects of Ganoderma lucidum polysaccharides on the proliferation of triple-negative breast cancer cells (MDA-MB-231) and breast cancer cells (MCF-7). Figures (A), (C), (E), and (G) respectively show the effects of Ganoderma lucidum crude polysaccharides GS1, GS2, GS3, and GS4 on the proliferation of MDA-MB-231 cells, and Figures (B), (D), (F), and (H) respectively show the effects of Ganoderma lucidum crude polysaccharides GS1, GS2, GS3, and GS4 on the proliferation of MCF-7 cells; Figures (I) and (J) respectively show the effects of Ganoderma lucidum polysaccharide GSP4-2 on the proliferation of MDA-MB-231 cells and MCF-7 cells.

[0095] Fig.10:The clone formation experiment of Ganoderma lucidum crude polysaccharide GS4 on MDA-MB-231 cells. Figures (A) and (B) show the inhibition of MDA-MB-231 cell proliferation by GS4 and its quantitative results, respectively.

[0096] Fig.11 :The clone formation experiment of GSP4-2 on MDA-MB-231 cells. Figures (A) and (B) show the proliferation inhibition of MDA-MB-231 cells by GSP4-2 and its quantitative results, respectively.

[0097] Fig.12 :The scratch test of Ganoderma lucidum crude polysaccharide GS4 on MDA-MB-231 cells. Figures (A) and (B) show the effect of GS4 on the migration ability of MDA-MB-231 cells and its quantitative results, respectively.

[0098] Fig.13 :The cell scratch test of GSP4-2, a polysaccharide of Ganoderma lucidum, on MDA-MB-231 cells. Figures (A) and (B) show the effect of GSP4-2 on the migration ability of MDA-MB-231 cells and its quantitative results, respectively.

[0099] Fig.14 :Invasion experiment of Ganoderma lucidum crude polysaccharide GS4 on MDA-MB-231 cells. Figures (A) and (B) show the effect of GS4 on the invasion ability of MDA-MB-231 cells and its quantitative results, respectively.

[0100] Fig.15 :Invasion experiment of GSP4-2, a polysaccharide from Ganoderma lucidum, on MDA-MB-231 cells. Figures (A) and (B) show the effect of GSP4-2 on the invasion ability of MDA-MB-231 cells and its quantitative results, respectively.

[0101] Fig.16 :Effects of Ganoderma lucidum crude polysaccharide GS4 on the expression of EMT-related genes Snail(A), ZEB1(B), VIM(C), CDH1(D), CDH2(E) and MMP9(F) in MDA-MB-231 cells.

[0102] Fig.17 : Effects of Ganoderma lucidum polysaccharide GSP4-2 on the expression of EMT-related genes Snail(A), ZEB1(B), VIM(C), CDH1(D), CDH2(E) and MMP9(F) in MDA-MB-231 cells.

[0103] Fig.18 :Effects of Ganoderma lucidum crude polysaccharide GS4 on (A) cell viability, (B) angiogenesis and (C) quantitative results of human umbilical vein endothelial cells (HUVECs).

[0104] Fig.19 :Effects of Ganoderma lucidum polysaccharide GSP4-2 on (A) cell viability, (B) angiogenesis and (C) quantitative results of human umbilical vein endothelial cells (HUVECs).

[0105] Fig. 20 :Effects of Ganoderma lucidum crude polysaccharides GS1, GS2, GS3 and GS4 on (A) tumor volume, (B) tumor weight, (C) tumor inhibition rate and (D) tumor volume in 4T1 tumor-bearing BALB / c mice.

[0106] Fig.21 : Effects of Ganoderma lucidum crude polysaccharides GS1, GS2, GS3 and GS4 on (A) body weight and organ indexes ((B) and (C)) of 4T1 tumor-bearing BALB / c mice.

[0107] Above Figure 7-Figure 19 Compared with the control group (Control), **P<0.001, **P<0.01, *P<0.05. DETAILED DESCRIPTION

[0108] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0109] Example 1 Preparation of Ganoderma lucidum polysaccharide

[0110] Prepare Ganoderma lucidum polysaccharide according to the following steps:

[0111] 1) Material selection: Cut 45.5 kg of dried Ganoderma lucidum fruiting bodies into small pieces of 2 to 5 cm and soak them in water for 10 hours;

[0112] 2) Water extraction: extract the Ganoderma lucidum fruiting body soaked in step 1) with hot water (80°C) at a solid-liquid ratio of 1:10 (w / v) for 4 hours, collect the extract and residue, and dry the residue;

[0113] 3) Graded alcohol precipitation: the extract obtained in step 2) is concentrated under reduced pressure at 50°C to one tenth of the original volume, ethanol is added for alcohol precipitation so that the volume concentration of ethanol is 55%, and the mixture is allowed to stand at room temperature for 24 hours, followed by centrifugation to obtain supernatant 1 and precipitate 1, and the precipitate 1 is collected as crude polysaccharide GS1; the supernatant 1 is concentrated under reduced pressure at 50°C, ethanol is added for alcohol precipitation so that the volume concentration of ethanol is 75%, and the mixture is allowed to stand at room temperature for 24 hours, followed by centrifugation to obtain supernatant 2 and precipitate 2, and the precipitate 2 is collected as crude polysaccharide GS2; the supernatant 2 is concentrated under reduced pressure at 50°C, ethanol is added for alcohol precipitation so that the volume concentration of ethanol is 95%, and the mixture is allowed to stand, followed by centrifugation to obtain precipitate 3, and the precipitate 3 is collected to obtain crude polysaccharide GS3;

[0114] 4) Alkali extraction: soak the residue obtained in step 2) in a 0.4M NaOH solution with a solid-liquid ratio of 1:10 (w / v), stand at room temperature for 3 hours to obtain supernatant three, neutralize supernatant three with 0.6M HCl to a pH value of 7.0, centrifuge (1768×g, 10 min) to obtain supernatant four, concentrate supernatant four at 50°C under reduced pressure, add ethanol for alcohol precipitation to make the ethanol volume concentration of 70%, stand at room temperature for 24 hours, centrifuge to obtain precipitate four, and collect the precipitate to be crude polysaccharide GS4;

[0115] 5) Protein removal: Use the Sevag method to remove protein from each crude polysaccharide, that is, add Sevag reagent (chloroform: n-butanol = 4:1) to the crude polysaccharide solution at a ratio of 5:1, shake vigorously and let it stand, remove the protein layer after the stratification is obvious, and repeat the steps 3-5 times. After protein removal, the crude polysaccharide is dialyzed with a dialysis bag (molecular weight cutoff of 1000Da) and freeze-dried to obtain the deproteinized Ganoderma lucidum crude polysaccharides GS1, GS2, GS3, and GS4;

[0116] 6) Ion exchange column chromatography: Fig. 9 It can be seen that among the four Ganoderma lucidum crude polysaccharides, GS4 has the best activity in inhibiting triple-negative breast cancer cells. Take 200 mg of Ganoderma lucidum crude polysaccharide GS4, dissolve it in 10 mL of deionized water, and load it on a DEAE-Cellulose 52 column. Four peaks appear under the conditions of different salt concentrations of eluents, of which Peak 1 is the 0M NaCl elution part, Peak 2 is the 0.05M NaCl elution part, Peak 3 is the 0.15M NaCl elution part, and Peak 4 is the 0.2M NaCl elution part (the elution curve is monitored by the phenol-sulfuric acid method during the elution process, and the sugar parts are collected separately according to the elution curve). The obtained eluents are concentrated under reduced pressure at 50°C and freeze-dried to obtain four polysaccharides (peak 1 polysaccharide, peak 2 polysaccharide, peak 3 polysaccharide, peak 4 polysaccharide);

[0117] 7) Molecular sieve gel chromatography:

[0118] The peak 1 polysaccharide sample (high polysaccharide content) obtained in step 6) is dissolved in water, and the supernatant is taken after centrifugation, applied to a Sephacryl S-100 column, eluted with water, and the elution curve is monitored by the phenol-sulfuric acid method. The main peak is collected, concentrated under reduced pressure at 50°C, and freeze-dried to obtain Ganoderma lucidum polysaccharides GSP4-1-2 and GSP4-1-3.

[0119] The peak 2 polysaccharide sample (high polysaccharide content) obtained in step 6) is dissolved in water, and the supernatant is taken after centrifugation, applied to a Sephacryl S-100 column, eluted with water, and the elution curve is monitored by the phenol-sulfuric acid method. The main peak is collected, concentrated under reduced pressure at 50°C, and freeze-dried to obtain the Ganoderma lucidum polysaccharide GSP4-2.

[0120] Purity detection: The Ganoderma lucidum polysaccharides GSP4-1-2, GSP4-1-3 and GSP4-2 were prepared into a 2% (W / V) aqueous solution, and the elution curve was measured by HPGPC method.

[0121] The components GSP4-1-2, GSP4-1-3, and GSP4-2 obtained after separation and purification by ion exchange and gel filtration showed single symmetrical peaks, indicating that GSP4-1-2, GSP4-1-3, and GSP4-2 were uniform Ganoderma lucidum polysaccharides.

[0122] Example 2

[0123] Prepare Ganoderma lucidum polysaccharide according to the following steps:

[0124] 1) Material selection: Cut 45.5 kg of dried Ganoderma lucidum fruiting bodies into small pieces of 2 to 5 cm and soak them in water for 6 hours;

[0125] 2) Water extraction: extract the Ganoderma lucidum fruiting body soaked in step 1) with hot water (100° C.) at a solid-liquid ratio of 1:5 (w / v) for 1 h, collect the extract and residue, and dry the residue;

[0126] 3) Graded alcohol precipitation: the extract obtained in step 2) is concentrated under reduced pressure at 70°C to one tenth of the original volume, ethanol is added for alcohol precipitation so that the volume concentration of ethanol is 20%, and the mixture is allowed to stand at room temperature for 10 hours, followed by centrifugation to obtain supernatant 1 and precipitate 1, and the precipitate 1 is collected as crude polysaccharide GS1; the supernatant 1 is concentrated under reduced pressure at 70°C, ethanol is added for alcohol precipitation so that the volume concentration of ethanol is 60%, and the mixture is allowed to stand at room temperature for 10 hours, followed by centrifugation to obtain supernatant 2 and precipitate 2, and the precipitate 2 is collected as crude polysaccharide GS2; the supernatant 2 is concentrated under reduced pressure at 70°C, ethanol is added for alcohol precipitation so that the volume concentration of ethanol is 80%, and the mixture is allowed to stand, followed by centrifugation to obtain precipitate 3, and the precipitate 3 is collected to obtain crude polysaccharide GS3;

[0127] 4) Alkaline extraction: soak the residue obtained in step 2) in a 0.1M NaOH solution with a solid-liquid ratio of 1:5 (w / v), stand at room temperature for 1 hour to obtain supernatant three, neutralize supernatant three with 0.1M HCl to a pH value of 7.0, centrifuge (1768×g, 10 min) to obtain supernatant four, concentrate supernatant four under reduced pressure at 70°C, add ethanol for alcohol precipitation to make the ethanol volume concentration be 30%, stand at room temperature for 10 hours, then centrifuge to obtain precipitate four, collect the precipitate to be crude polysaccharide GS4;

[0128] 5) Protein removal: Use the Sevag method to remove protein from each crude polysaccharide, that is, add Sevag reagent (chloroform: n-butanol = 4:1) to the crude polysaccharide solution at a ratio of 5:1, shake vigorously and let it stand, remove the protein layer after the stratification is obvious, and repeat the steps 3-5 times. After protein removal, the crude polysaccharide is dialyzed with a dialysis bag (molecular weight cutoff of 1000Da) and freeze-dried to obtain Ganoderma lucidum crude polysaccharides GS1, GS2, GS3, and GS4;

[0129] 6) Ion exchange column chromatography: 200 mg of the purified Ganoderma lucidum crude polysaccharide GS4 was taken, dissolved in 10 mL of deionized water, and loaded on a DEAE-Cellulose 52 column. Four peaks appeared under the conditions of different salt concentrations of the eluent, wherein Peak 1 was the 0 M NaCl elution portion, Peak 2 was the 0.05 M NaCl elution portion, Peak 3 was the 0.15 M NaCl elution portion, and Peak 4 was the 0.2 M NaCl elution portion (the elution curve was tracked by the phenol-sulfuric acid method during the elution process, and the sugar portions were collected separately according to the elution curve). The obtained eluents were concentrated under reduced pressure at 70° C. and freeze-dried to obtain four polysaccharides (peak 1 polysaccharide, peak 2 polysaccharide, peak 3 polysaccharide, peak 4 polysaccharide);

[0130] 7) Molecular sieve gel chromatography:

[0131] The peak 1 polysaccharide sample (high polysaccharide content) obtained in step 6) is dissolved in water, and the supernatant is taken after centrifugation, applied to a Sephacryl S-100 column, eluted with water, and the elution curve is monitored by the phenol-sulfuric acid method. The main peak is collected, concentrated under reduced pressure at 50°C, and freeze-dried to obtain Ganoderma lucidum polysaccharides GSP4-1-2 and GSP4-1-3.

[0132] The peak 2 polysaccharide sample (high polysaccharide content) obtained in step 6) is dissolved in water, and the supernatant is taken after centrifugation, applied to a Sephacryl S-100 column, eluted with water, and the elution curve is monitored by the phenol-sulfuric acid method. The main peak is collected, concentrated under reduced pressure at 50°C, and freeze-dried to obtain the Ganoderma lucidum polysaccharide GSP4-2.

[0133] Example 3

[0134] Prepare Ganoderma lucidum polysaccharide according to the following steps:

[0135] 1) Material selection: Soak 45.5 kg of dried Ganoderma lucidum fruiting bodies in water for 8 hours;

[0136] 2) Water extraction: extract the Ganoderma lucidum fruiting body soaked in step 1) with hot water (80°C) at a solid-liquid ratio of 1:15 (w / v) for 10 hours, collect the extract and residue, and dry the residue;

[0137] 3) Graded alcohol precipitation: the extract obtained in step 2) is concentrated under reduced pressure at 40°C to one tenth of the original volume, ethanol is added for alcohol precipitation so that the volume concentration of ethanol is 59%, and the mixture is allowed to stand at room temperature for 28 hours, followed by centrifugation to obtain supernatant 1 and precipitate 1, and the precipitate 1 is collected as crude polysaccharide GS1; the supernatant 1 is concentrated under reduced pressure at 40°C, ethanol is added for alcohol precipitation so that the volume concentration of ethanol is 79%, and the mixture is allowed to stand at room temperature for 28 hours, followed by centrifugation to obtain supernatant 2 and precipitate 2, and the precipitate 2 is collected as crude polysaccharide GS2; the supernatant 2 is concentrated under reduced pressure at 40°C, ethanol is added for alcohol precipitation so that the volume concentration of ethanol is 99%, and the mixture is allowed to stand, followed by centrifugation to obtain precipitate 3, and the precipitate 3 is collected to obtain crude polysaccharide GS3;

[0138] 4) Alkali extraction: soak the residue obtained in step 2) in a 1M NaOH solution with a solid-liquid ratio of 1:15 (w / v), stand at room temperature for 4 hours to obtain supernatant three, neutralize supernatant three with 1M HCl to a pH value of 7.0, centrifuge (1768×g, 10 min) to obtain supernatant four, concentrate supernatant four under reduced pressure at 40°C, add ethanol for alcohol precipitation to make the ethanol volume concentration be 90%, stand at room temperature for 28 hours, then centrifuge to obtain precipitate four, collect the precipitate to be crude polysaccharide GS4;

[0139] 5) Purification: Use the Sevag method to remove protein from each crude polysaccharide, that is, add Sevag reagent (chloroform: n-butanol = 4:1) to the crude polysaccharide solution at a ratio of 5:1, shake vigorously and let it stand, remove the protein layer after the stratification is obvious, and repeat the steps 3-5 times. After removing the protein, the crude polysaccharide is dialyzed with a dialysis bag (molecular weight cutoff of 1000Da) and freeze-dried to obtain Ganoderma lucidum crude polysaccharides GS1, GS2, GS3, and GS4;

[0140] 6) Ion exchange column chromatography: 200 mg of the purified Ganoderma lucidum crude polysaccharide GS4 was taken, dissolved in 10 mL of deionized water, and loaded on a DEAE-Cellulose 52 column. Four peaks appeared under the conditions of different salt concentrations of the eluent, wherein Peak 1 was the 0 M NaCl elution portion, Peak 2 was the 0.05 M NaCl elution portion, Peak 3 was the 0.15 M NaCl elution portion, and Peak 4 was the 0.2 M NaCl elution portion (the elution curve was tracked by the phenol-sulfuric acid method during the elution process, and the sugar portions were collected separately according to the elution curve). The obtained eluents were concentrated under reduced pressure at 70° C. and freeze-dried to obtain four polysaccharides (peak 1 polysaccharide, peak 2 polysaccharide, peak 3 polysaccharide, peak 4 polysaccharide);

[0141] 7) Molecular sieve gel chromatography: The peak 1 polysaccharide sample (high polysaccharide content) obtained in step 6) is dissolved in water, centrifuged and separated, the supernatant is collected, applied to a Sephacryl S-100 column, eluted with water, the elution curve is monitored by the phenol-sulfuric acid method, the main peak is collected, concentrated under reduced pressure at 50°C, and freeze-dried to obtain Ganoderma lucidum polysaccharides GSP4-1-2 and GSP4-1-3.

[0142] The peak 2 polysaccharide sample (high polysaccharide content) obtained in step 6) is dissolved in water, and the supernatant is taken after centrifugation, applied to a Sephacryl S-100 column, eluted with water, and the elution curve is monitored by the phenol-sulfuric acid method. The main peak is collected, concentrated under reduced pressure at 50°C, and freeze-dried to obtain the Ganoderma lucidum polysaccharide GSP4-2.

[0143] Example 4

[0144] The following is a further structural analysis of the GSP4-2 extracted and separated in Example 1:

[0145] (1) Purity verification

[0146] The HPGPC method showed that GSP4-2 was a uniform polysaccharide with a single symmetrical peak.

[0147] (2) Monosaccharide composition analysis

[0148] The spectrum of the complete acid hydrolysis product measured by PMP-pre-column derivatization high performance liquid chromatography showed that GSP4-2 is an acidic heteropolysaccharide composed of mannose, glucuronic acid, glucose, galactose, and fucose in a ratio of 8.0:3.2:19.7:4.3:1.0.

[0149] (3) Infrared spectrum detection

[0150] The infrared spectrum of GSP4-2 (such as Figure 1 The test results (shown) showed that GSP4-2 contained characteristic absorption peaks of sugar.

[0151] (4) Methylation analysis

[0152] The samples were subjected to uronic acid reduction, methylation, hydrolysis, reduction, and acetylation, and then GC-MS analysis. The results showed that GSP4-2 contained L-Fucp-(1→, D-Galp-(1→, D-GalpA-(1→, →3)-D-Glcp-(1→, →3)-D-Glcp-(1→, →4)-D-Galp-(1→, →6)-D-Manp-(1→ and →3,6)-D-Glcp-(1→ sugar residues.

[0153] (5) Nuclear magnetic resonance analysis of polysaccharides

[0154] The homogeneous refined polysaccharide GSP4-2 sample was placed in a nuclear magnetic resonance tube, dissolved in D2O and then measured, and deuterated acetone was used as an internal standard (δ C 215.9) The result is as follows Figure 2 to Figure 6 shown.

[0155] According to the above Figure 2 to Figure 6 The NMR spectrum shows that each 1 H and 13 C data attribution is shown in Table 1.

[0156] Table 1 GSP4-2 sugar residues 1 H and 13 C Data Ownership

[0157]

[0158]

[0159] The results of the above-mentioned complete acid hydrolysis, methylation analysis, infrared spectroscopy and nuclear magnetic resonance analysis showed that GSP4-2 is an acidic heteropolysaccharide composed of mannose, glucuronic acid, glucose, galactose, and fucose, with a molar ratio of 8.0:3.2:19.7:4.3:1.0. The methylation analysis showed that it contained L-Fucp-(1→, D-Galp-(1→, D-GalpA-(1→, →3)-D-Glcp-(1→, →3)-D-Glcp-(1→, →4)-D-Galp-(1→, →6)-D-Manp-(1→ and →3,6)-D-Glcp-(1→ sugar residues. The connection sequence between different sugar residues was obtained by two-dimensional nuclear magnetic resonance HMBC spectrum analysis. From the above analysis, the structure of GSP4-2 with a molecular weight range of 10k to 200k Da is as follows:

[0160]

[0161] Among them, a, b, c, d, and e range from 1 to 100;

[0162] Similarly, the GSP4-1-2 and GSP4-1-3 extracted and separated in Example 1 were analyzed in the same manner as above to obtain their structures.

[0163] The structural formula of Ganoderma lucidum polysaccharide GSP4-1-2 (see NMR spectrum) Figure 7 ) is as follows:

[0164]

[0165] Among them, the range of a, b, c, d, and e is 1 to 100.

[0166] The molecular weight of GSP4-1-2 is in the range of 7k to 100k Da; GSP4-1-2 includes mannose, glucose, galactose, xylose and fucose; the molar ratio is 21.1:16.3:51.2:4.1:8.2.

[0167] The structural formula of Ganoderma lucidum polysaccharide GSP4-1-3 (see NMR spectrum) Figure 8 ) is as follows:

[0168]

[0169] Among them, the range of a, b, c, d, and e is 1 to 100.

[0170] The molecular weight of GSP4-1-3 is in the range of 10k to 200k Da; GSP4-1-3 includes mannose, glucose, galactose and fucose; and the molar ratio is 5.4:4.1:42.8:5.4.

[0171] The above-mentioned uniform polysaccharides may be the active ingredients of Ganoderma lucidum in selectively inhibiting the proliferation, invasion and metastasis of triple-negative breast cancer cells. Therefore, the structural identification of uniform polysaccharides will provide a strong basis for subsequent research on the mechanism by which Ganoderma lucidum selectively inhibits the proliferation, invasion and metastasis of triple-negative breast cancer cells.

[0172] Example 5: Study on the inhibition of triple-negative breast cancer cell proliferation by Ganoderma lucidum crude polysaccharide GS4 and refined polysaccharide GSP4-2

[0173] 1. MTT colorimetric method: Take logarithmic phase MDA-MB-231 cells and adjust the cell density to 5×10 4 / mL; 100 μL of cell suspension per well was inoculated into a 96-well plate and incubated in an incubator for 24 hours; the old culture medium in the 96-well plate was removed, and different concentrations of drug-containing culture medium were added, 100 μL / well, and cultured for 48 hours (at least 3 replicates were set for each concentration), wherein the drug-treated group included: 100, 200, 400, 800, 1600 μg / mL of GS1, GS2, GS3 and GS4 in Example 1; 25, 50, 100, 200, 400 μM of GSP4-2; DDP (10 μM); the control group cells were added with the same volume of DMEM culture medium containing 10% FBS and 1% penicillin-streptomycin; 10 μL of 5 mg / mL MTT solution was added to the drug-treated group and the control group, respectively, and the blank group was not added with MTT solution, and was placed in an incubator for further culture for 4 hours; the staining solution was carefully removed to avoid absorbing formazan, and 100 μL of DMSO was added and shaken to completely dissolve the formazan. The result was detected by an ELISA reader (490 nm). The cell growth curve was drawn with the concentration as the horizontal axis and the cell survival rate as the vertical axis.

[0174] Calculation formula:

[0175] The results are as follows Fig. 9 (A)- Fig. 9 As shown in (H), the crude polysaccharides GS1, GS2, GS3 and GS4 of Ganoderma lucidum were able to effectively inhibit the proliferation of MDA-MB-231 cells after acting on MDA-MB-231 cells for 48 hours, compared with the Control group, and showed corresponding concentration dependence (400-1600μg / mL, P<0.001), while GS1, GS2, GS3 and GS4 had no significant inhibitory activity compared with the Control group after acting on MCF-7 cells for 48 hours. Among them, GS4 began to inhibit MDA-MB-231 cells at 100μg / mL (P<0.05), and was concentration-dependent. Therefore, the crude polysaccharide GS4 of Ganoderma lucidum was separated and purified to obtain the Ganoderma lucidum polysaccharide GSP4-2.

[0176] Effects of Ganoderma lucidum polysaccharide GSP4-2 on the proliferation of MDA-MB-231 cells and MCF-7 cells Fig. 9 (I)- Fig. 9As shown in (J), GSP4-2 only exhibited a concentration-dependent proliferation inhibitory effect (50-400 μM) on MDA-MB-231 cells, and had no inhibitory activity on MCF-7 cells. Therefore, the crude polysaccharides GS1, GS2, GS3 and GS4 of Ganoderma lucidum and the refined polysaccharide GSP4-2 had selective inhibitory activity on the proliferation of MDA-MB-231 cells in in vitro cell experiments. Among them, the crude polysaccharide GS4 had the best inhibitory effect on the proliferation of MDA-MB-231, so the crude polysaccharide GS4 of Ganoderma lucidum and the refined polysaccharide GSP4-2 obtained by separation and purification were selected to perform related experiments on the inhibition of proliferation, invasion and metastasis of triple-negative breast cancer cells MDA-MB-231.

[0177] 2. Cell clone formation experiment: Take MDA-MB-231 cells in the logarithmic growth phase and adjust the cell density to 2.0×10 2 / mL, inoculated in a 24-well plate, placed in an incubator for incubation for 24 hours before administration; set up a Control group, a GS4 low concentration group (50μg / mL), a GS4 medium concentration group (100μg / mL), a GS4 high concentration group (200μg / mL), a GSP4-2 low concentration group (50μM), a GSP4-2 medium concentration group (100μM), and a GSP4-2 high concentration group (200μM). Each group was set up with three replicates. After 48 hours of administration, the drug-containing culture medium was discarded, and a DMEM culture medium containing 10% FBS was added. The culture was continued for 10 days and observed every day. On the 10th to 12th day, the 24-well plate was removed, fixed with 4% paraformaldehyde for 10 minutes, and then stained with 0.1% crystal violet for 20 minutes (at room temperature). The dye solution was gently washed off with PBS, photographed and observed, and data statistics were performed using prism software.

[0178] The experimental results of crude polysaccharide GS4 are as follows Fig.10 (A)- Fig.10 As shown in (B), the cell colonies in the control group were dense, while the number of cell colonies formed after GS4 treatment was less and smaller than that in the control group (P<0.05), and showed a certain concentration dependence. Fig.11 (A) and Fig.11 As shown in (B), the cell colonies treated with GSP4-2 formed fewer and smaller colonies than those in the Control group, showing a certain concentration dependence. Among them, GSBP-2 inhibited the formation of MDA-MB-231 cell colonies most significantly at 200 μM (P<0.001). This suggests that both the crude polysaccharide GS4 and the refined polysaccharide GSP4-2 of Ganoderma lucidum can inhibit the proliferation of MDA-MB-231 cells.

[0179] The crude polysaccharides GS1, GS2, GS3 and GS4 extracted from Ganoderma lucidum can selectively inhibit the proliferation of MDA-MB-231 cells. Among them, the refined polysaccharide GSP4-2 obtained by separation and purification of GS4 may be the effective component that exerts the above-mentioned activity, and has the activity of significantly selectively inhibiting the proliferation of human triple-negative breast cancer cells (MDA-MB-231). Its mechanism of action against MDA-MB-231 cells will be further studied in subsequent experiments.

[0180] Example 6: Study on the inhibitory activity of crude polysaccharide GS4 and refined polysaccharide GSP4-2 from Ganoderma lucidum on the migration and invasion of MDA-MB-231 cells

[0181] 1. Scratch test: (1) Scratch the culture plate. Use a marker pen and a ruler to evenly draw horizontal lines on the back of the 6-well plate, approximately every 0.5 to 1 cm, across the wells. Make at least 5 lines in each well. (2) Add 2 mL of 2×10 5 / mL of MDA-MB-231 cell suspension; (3) The next day, use a 200μL pipette tip, perpendicular to the cell plane, to scratch the cell layer along the drawn line (it is best to use the same pipette tip between different wells); (4) After the scratching is completed, wash the cells three times with sterile PBS to wash away dead cells that are not attached to the wall, so that the gap left after the scratching is clearly visible. Then replace with fresh serum-free culture medium and sample solutions of different concentrations; (5) Place the cells in a CO2 incubator for culture. Take out the cells at 0, 12 and 24 hours, observe and measure the width of the scratch under a microscope, and take pictures to record (6) After opening the picture using Image J software, randomly select at least 5 points and calculate the distance between cells.

[0182] The qualitative and quantitative experimental results of Ganoderma lucidum crude polysaccharide GS4 are as follows Fig.12 (A)- Fig.12 As shown in (B), in the Control group, the damaged MDA-MB-231 cell layer showed a healing trend over time within 24 hours, indicating that MDA-MB-231 cells have strong motility under normal conditions. The GS4 group showed a strong inhibitory effect on wound healing (P<0.05), and it was concentration-dependent. Among them, the wound healing of the 200μg / mL GS4 group was significantly different from that of the Control group after 12 hours. The qualitative and quantitative experimental results of Ganoderma lucidum polysaccharide GSP4-2 are shown in Figure 2. Fig.13 (A)- Fig.13As shown in (B), compared with the Control group, the GSP4-2 group showed a certain inhibition on wound healing, and it was concentration-dependent. Among them, 200μM GSP4-2 treatment for 24h had the strongest inhibitory effect on wound healing (P<0.01). The above results show that both the crude polysaccharide GS4 and the refined polysaccharide GSP4-2 of Ganoderma lucidum have the ability to inhibit the migration of MDA-MB-231 cells.

[0183] 2. Transwell invasion assay: (1) Transfer the aliquoted Matrigel from -20°C to a 4°C refrigerator overnight, and the Matrigel will melt from a solid state to a liquid state; (2) Dilute the Matrigel: medium at a ratio of 1:2 or 1:3, coat the upper chamber surface of the bottom membrane of the transwell chamber, and place in an incubator to wait for it to solidify; (3) Wash with PBS 1 to 2 times to remove the influence of serum, digest with trypsin, resuspend the MDA-MB-231 cells in serum-free medium, and adjust the cell density to 2×10 4 / mL; (4) Take 100μL of cell suspension and add it to the upper chamber of Transwell, add 100μL of polysaccharide sample solution of different concentrations, and add 600μL of 10% FBS culture medium to the lower chamber of 24-well culture plate. (5) The culture plate is placed in a CO2 incubator and cultured for 12 to 48h; (6) Take out the chamber, wash it twice with PBS, wipe the cells in the upper layer of the microporous membrane of the chamber with a cotton swab, fix it with 4% paraformaldehyde in a 24-well plate for 20min, wash it three times with PBS, and stain it with crystal violet solution for 15min; (7) After washing with PBS, take pictures under an inverted microscope, randomly count 10 fields of view for each sample, take the average value, and perform statistical analysis.

[0184] The qualitative and quantitative experimental results of Ganoderma lucidum crude polysaccharide GS4 are as follows Fig.14 (A)- Fig.14 As shown in (B), the number of MDA-MB-231 cells that passed through the matrix capsule in the Control group was relatively large, indicating that the invasion of MDA-MB-231 cells did exist. After 24 hours of GS4 treatment, the number of invasion of MDA-MB-231 cells was significantly reduced (P<0.001). With the increase of sample concentration, GS4 had a significant inhibitory effect on the invasion ability of cells. The qualitative and quantitative experimental results of GSP4-2 polysaccharide from Ganoderma lucidum are shown in Figure 2. Fig.15 (A)- Fig.15 As shown in (B), compared with the Control group, the number of MDA-MB-231 cells treated with GSP4-2 for 24 hours was significantly reduced in invasion, and the number of cells passing through the matrix gel was significantly reduced at 100μM and 200μM concentrations (P<0.001). The above results show that both Ganoderma lucidum crude polysaccharide GS4 and Ganoderma lucidum fine polysaccharide GSP4-2 have the ability to inhibit the invasion of MDA-MB-231 cells.

[0185] 3. qPCR experiment: qPCR was used to analyze the effects of Ganoderma lucidum crude polysaccharide GS4 and refined polysaccharide GSP4-2 on the transcription levels of EMT-related genes in MDA-MB-231 cells.

[0186] The primer designs are shown in the following table:

[0187] Table 2 qRT-PCR primer sequences

[0188]

[0189] The results are as follows Figure 16-17 As shown. Snail1, ZEB1, VIM, CDH2 and MMP9 genes, which are positively correlated with EMT, showed concentration-dependent inhibition of expression after treatment with Ganoderma lucidum crude polysaccharide GS4 and GSP4-2, while CDH1 gene, which is negatively correlated with EMT, showed concentration-dependent promotion of expression. After treatment with 100μM GSP4-2, the expression of positively correlated genes was significantly decreased (P<0.001), while the expression of negatively correlated gene CDH1 was significantly increased after treatment with 200μM GSP4-2 (P<0.001), which was concentration-dependent. This suggests that Ganoderma lucidum crude polysaccharide GS4 and GSP4-2 specifically inhibit the transcriptional levels of EMT-related genes in MDA-MB-231 cells, thereby significantly inhibiting EMT-mediated invasion and metastasis.

[0190] Example 7: Effects of Ganoderma lucidum crude polysaccharide GS4 and refined polysaccharide GSP4-2 on angiogenesis

[0191] First, the toxicity of crude polysaccharide GS4 and refined polysaccharide GSP4-2 on HUVECs cells was detected by MTT colorimetry. Fig.18 (A) and Fig.19 As shown in (A), GS4 and GSP4-2 had no cytotoxic activity against HUVECs within the selected concentration range.

[0192] Angiogenesis experiment: (1) Place Matrigel and 96-well plate on ice, use a pre-cooled pipette tip to take 50 μL Matrigel into the 96-well plate and spread it evenly; (2) Place the 96-well plate in a 37°C incubator for 30 min to allow gelation; (3) Collect HUVEC cells and adjust the density to 6×10 5 / 100μL. The 96-well plate was taken out from the incubator. The experimental group was given 50μL of polysaccharide solution of different concentrations and 50μL of cell suspension, while the control group was given 50μL of 1640 culture medium and 50μL of cell suspension. Three replicate wells were set for each concentration; (4) The 96-well plate was placed in the incubator for 12h and waited for tube formation; (5) The 96-well plate was taken out, the upper culture medium was aspirated, 4% paraformaldehyde was added for fixation for 20min, and then washed with PBS, photographed under a microscope, and data processed using ImageJ.

[0193] The results of the angiogenesis experiment of Ganoderma lucidum crude polysaccharide GS4 and refined polysaccharide GSP4-2 are as follows Fig.18 As shown in Figures 18(B), 19(B), 19(C), GS4 significantly inhibited the formation of HUVECs blood vessels in a concentration-dependent manner (50-200 μg / mL) compared with the Control group (P<0.001). GSP4-2 significantly inhibited the formation of HUVECs blood vessels in a concentration-dependent manner (P<0.001). Therefore, both the crude polysaccharide GS4 and the refined polysaccharide GSP4-2 of Ganoderma lucidum can inhibit angiogenesis.

[0194] Example 8: In vivo anti-breast cancer experiment of Ganoderma lucidum crude polysaccharides GS1, GS2, GS3 and GS4

[0195] 1. Construct a BALB / c mouse model of 4T1 breast tumors, and randomly divide 42 tumor-bearing mice into a model (Control) group (equal volume of normal saline), a positive control group (cisplatin, intraperitoneal injection of 3 mg / kg, once every three days, for 14 days) and a Ganoderma lucidum crude polysaccharide group (GS1, GS2, GS3 and GS4, except for GS4 set at 200 mg / kg and 400 mg / kg, the other concentrations are 400 mg / kg, gavage 0.1 mL / 10 g, once a day), with 6 animals in each group. During the experiment, the animal response and body weight were recorded every other day, and the tumor volume was measured = (length × width 2 ) / 2; 14 days after the polysaccharide group was administered, the mice were anesthetized and killed, and the transplanted tumors and organs were dissected and removed. The weights were recorded and photographed. Organ index = organ weight / mouse body weight.

[0196] The results showed that the effects of Ganoderma lucidum crude polysaccharides (GS1, GS2, GS3 and GS4) and cisplatin on tumor volume and weight were as follows: Fig. 20As shown in (A) and 20(B), the transplanted tumor volume in the model (Control) group was large, indicating that the 4T1 transplanted tumor was successfully inoculated. The transplanted tumor volume of the Cisplatin group was significantly reduced 14 days after administration, indicating that it effectively inhibited the growth of the 4T1 tumor. In the polysaccharide administration group, the tumor volume of the GS2 and GS4 (400 mg / kg) groups was significantly reduced, showing a tumor inhibition effect close to that of the Cisplatin group; in terms of transplanted tumor weight, compared with the Control group, the tumor weight of the Cisplatin group, GS2 and GS4 (400 mg / kg) groups was significantly reduced (P < 0.001), and the tumor weight of the GS3 group was significantly reduced (P < 0.05). The tumor inhibition rate results are shown in Fig. 20 As shown in (C), the tumor inhibition rate of Cisplatin group was 69.03%, and the tumor inhibition rates of GS1, GS2, GS3 and GS4 (200 mg / kg and 400 mg / kg) groups were 21.04%, 37.51%, 16.05%, 14.63% and 29.38%, respectively; the changes in tumor volume of tumor-bearing BALB / c mice within 14 days are shown in Fig. 20 As shown in (D), compared with the Control group, the Cisplatin group had no obvious effect on the transplanted tumor at the beginning of administration. As the administration time prolonged, the inhibitory effect of the Cisplatin group on the transplanted tumor volume gradually became prominent (P < 0.001). As the administration time prolonged, GS2 and GS4 (400 mg / kg) in the Ganoderma lucidum crude polysaccharide group showed a certain inhibitory effect on the transplanted tumor volume (P < 0.001, P < 0.01). In summary, Ganoderma lucidum crude polysaccharides GS2, GS3 and GS4 have certain in vivo anti-breast cancer effects, among which GS2 and GS4 (400 mg / kg) have significant effects.

[0197] like Fig.21 As shown, the weight of mice in each group of Ganoderma lucidum crude polysaccharide did not decrease significantly, and the indexes of various organs did not change significantly, and there was no significant difference compared with the Control group, indicating that there was no obvious toxicity to mice. However, the weight of the cisplatin group decreased significantly (P < 0.001), and the liver index, spleen index, kidney index and lung index decreased significantly compared with the Control group (P < 0.001, P < 0.01), indicating that cisplatin has toxic side effects on mice. In summary, Ganoderma lucidum crude polysaccharides GS1, GS2, GS3 and GS4 had no significant effect on the weight and organ indexes of 4T1 tumor-bearing BALB / c mice, that is, no toxic side effects.

[0198] The above results show that Ganoderma lucidum crude polysaccharides GS2, GS3 and GS4 have certain anti-breast cancer effects in vivo, among which GS2 and GS4 can significantly inhibit the growth of breast cancer in vivo. Cisplatin, as a traditional chemotherapy drug, has toxic side effects on the body. However, Ganoderma lucidum crude polysaccharides can fight breast cancer without producing obvious toxic side effects and causing significant damage to the body, which means that they may have a protective effect. Therefore, it provides a basis for the application of Ganoderma lucidum polysaccharides as candidate drugs for the specific inhibition of triple-negative breast cancer or as auxiliary therapeutic drugs to improve the quality of life of patients.

[0199] Traditional surgical treatment for breast cancer, especially for triple-negative breast cancer with a strong tendency to invade and metastasize, is not effective, and there is still a possibility of postoperative recurrence. The vast majority of breast cancer-related deaths are related to its metastatic recurrence. Metastatic breast cancer can quickly lead to multiple organ failure, increasing the difficulty of treatment. The migration and invasion process of breast cancer is closely related to EMT. Therefore, inhibiting the expression of EMT-related genes in breast cancer cells themselves and inhibiting their promotion of angiogenesis are research hotspots and important strategies to prolong the survival of breast cancer patients after surgical treatment. The Ganoderma lucidum polysaccharide extracted by the above method shows selective inhibitory effects on triple-negative breast cancer cells, inhibitory effects on invasion and metastasis regulated by EMT, and inhibitory effects on angiogenesis. Therefore, the study of the activity of Ganoderma lucidum polysaccharide in the preparation of a selective inhibitory effect on the proliferation, invasion and metastasis of triple-negative breast cancer cells provides a basis for the application of Ganoderma lucidum polysaccharide in the medical field.

[0200] The above specific implementations have been described in detail for the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A ganoderma lucidum polysaccharide, comprising any one of GSP4-2, GSP4-1-2 and GSP4-1-3, characterized in that: The structural formula of the Ganoderma lucidum polysaccharide GSP4-2 is shown in formula (I): Among them, a, b, c, d, and e range from 1 to 100; Formula (I); The structural formula of the Ganoderma lucidum polysaccharide GSP4-1-2 is shown in formula (II): Among them, a, b, c, d, and e range from 1 to 100; Formula (II) The structural formula of the Ganoderma lucidum polysaccharide GSP4-1-3 is shown in formula (III): Among them, a, b, c, d, and e range from 1 to 100; Formula (III).

2. The Ganoderma lucidum polysaccharide according to claim 1, characterized in that The molecular weight range of the Ganoderma lucidum polysaccharide GSP4-2 is 10k to 200k Da; the molecular weight range of the Ganoderma lucidum polysaccharide GSP4-1-2 is 7k to 100k Da; the molecular weight range of the Ganoderma lucidum polysaccharide GSP4-1-3 is 10k to 200k Da.

3. A method for preparing the Ganoderma lucidum polysaccharide according to claim 1 or 2, comprising the following steps: S21: extracting Ganoderma lucidum with water and collecting the obtained residue; S22: extracting the residue obtained in step S21 with alkali to obtain crude polysaccharide GS4; S23: removing the protein in the crude polysaccharide GS4 described in step S22 to obtain the crude polysaccharide GS4 of Ganoderma lucidum; The crude polysaccharide GS4 of Ganoderma lucidum is further purified to obtain the GSP4-2, GSP4-1-2 and GSP4-1-3; The purification includes ion exchange chromatography and molecular sieve gel chromatography; The ion exchange chromatography includes ion exchange cellulose or ion exchange gel; the ion exchange chromatography uses 0-2M NaCl solution for gradient elution; the molecular sieve gel chromatography includes Sephadex G and Sephacryl S series molecular sieve chromatographic columns; the molecular sieve gel chromatography uses water for elution.

4. The preparation method according to claim 3, characterized in that: The specific operation of water extraction in step S21 includes: mixing Ganoderma lucidum with water, extracting at 60-100° C. for 1-10 hours, and the volume ratio of Ganoderma lucidum to water is 1:(5-15).

5. The preparation method according to claim 3, characterized in that: The specific operation of step S22 includes: soaking the residue described in step S21 in 0.1~1 M NaOH solution for alkali extraction, collecting the alkali extract, and adjusting the alkali extract to a pH of 6-8, obtaining a supernatant after solid-liquid separation, and precipitating the supernatant with alcohol to obtain crude polysaccharide GS4.

6. The preparation method according to claim 5, characterized in that: The volume ratio of the residue to the sodium hydroxide solution is 1:(5-20); the conditions for the alkali extraction include: standing at room temperature for 1-4 hours; the solid-liquid separation method includes centrifugation; the volume concentration of ethanol in the alcohol precipitation is 30-90%.

7. Use of the Ganoderma lucidum polysaccharide according to any one of claims 1 to 2 in the preparation of a drug; the Ganoderma lucidum polysaccharide is GSP4-2, and the drug is used to inhibit the proliferation, invasion, and metastasis of triple-negative breast cancer cells, prevent and / or treat triple-negative breast cancer, and inhibit angiogenesis.

8. Use of the Ganoderma lucidum polysaccharide according to any one of claims 1 to 2 in the preparation of a drug; the Ganoderma lucidum polysaccharide is GSP4-1-2 or GSP4-1-3, and the drug is used to inhibit the proliferation of triple-negative breast cancer cells.

9. A medicine, characterized in that: The medicine contains the Ganoderma lucidum polysaccharide according to any one of claims 1 to 2.

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