Tremella polysaccharide prepared by using a eutectic solvent and application thereof in preparation of anti-breast cancer drugs

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a tremella polysaccharide prepared by a low eutectic solvent method and application thereof in preparation of an anti-breast cancer drug. The present application first discovers that the tremella polysaccharide extracted by the low eutectic solvent method has a significant effect of treating breast cancer, and can resist breast cancer through inhibition of cell proliferation, inhibition of cell migration and invasion and other pathways. Compared with the tremella polysaccharide obtained by a traditional water extraction and alcohol precipitation method, the tremella polysaccharide obtained by the low eutectic solvent method has a lower total sugar content and weight average molecular weight, and a slightly higher protein content, and the tremella polysaccharide can not only significantly improve inhibition of breast cancer cell proliferation, but also significantly inhibit migration and invasion of breast cancer cells. This multi-target effect mode can effectively block the source of tumor diffusion, improve the treatment effect and the survival rate of patients, reduce the risk of recurrence and metastasis, and enhance the overall effect of treatment, and has a good application prospect in the treatment of breast cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More specifically, it relates to Tremella polysaccharides prepared by a deep eutectic solvent method and the application thereof in preparing anti-breast cancer drugs. BACKGROUND

[0002] In recent years, with the continuous deepening of biotechnology and natural product research, scientists have gradually turned their attention to the excavation and application of traditional Chinese medicinal materials and their active ingredients. Tremella, as a fungus food with a long history and a medicinal and edible fungus, has attracted much attention due to its rich nutritional components and potential medicinal value. Tremella polysaccharides (TFPs), as one of the main active ingredients in Tremella, have shown great application prospects in the fields of medicine, health care and functional food due to their unique biological activity and good biocompatibility.

[0003] Deep eutectic solvents (DESs), as a new type of green solvent system, have attracted widespread attention in the field of natural product extraction in recent years due to their simple preparation, low cost, biodegradability and good solubility for a variety of substances. Compared with traditional organic solvents, deep eutectic solvents not only effectively retain the structure and biological activity of the target active ingredients during the extraction process, but also reduce environmental pollution, making them an environmentally friendly extraction solvent.

[0004] In the prior art, there have been studies on the extraction of active ingredients, including polysaccharide compounds, from various plants and microorganisms using deep eutectic solvents. For example, Chinese patent application CN113354754A discloses a method for extracting Tremella polysaccharides from Tremella spore fermentation broth using a deep eutectic solvent. This method effectively improves the extraction efficiency and purity of Tremella polysaccharides by optimizing the solvent composition and extraction conditions. However, this patent only focuses on the extraction process itself and does not delve into or disclose the specific biological activity and potential applications of the extracted Tremella polysaccharides, particularly their potential value in medical treatment.

[0005] In addition, although a large number of literature reports have shown that Tremella polysaccharides have various biological activities such as antioxidant, immunomodulatory and anti-tumor activities, there is still a lack of systematic research and clear evidence on their therapeutic effects on a specific disease such as breast cancer, especially on the inhibition of their migration and invasion ability. Moreover, there are few reports on the significant improvement of anti-tumor activity of Tremella polysaccharides extracted by deep eutectic solvents. This research gap not only limits the further development of Tremella polysaccharides in clinical applications, but also provides a space for exploration and innovation for researchers.

[0006] In summary, although the prior art has made some progress in the extraction method of tremella polysaccharide and its general biological activity, it is still insufficient in the research on the therapeutic effect for specific diseases. Therefore, it has important scientific significance and clinical application value to develop new extraction technology and further explore the potential of tremella polysaccharide in disease treatment, especially its inhibitory effect on specific tumors (such as breast cancer). SUMMARY

[0007] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the prior art focusing on the extraction process itself, without in-depth exploration or disclosure of the specific biological activity and potential application of the extracted tremella polysaccharide, and to provide the application of tremella polysaccharide in preparing anti-breast cancer drugs.

[0008] The purpose of the present application is to provide the application of tremella polysaccharide in preparing drugs for inhibiting the proliferation of breast cancer.

[0009] Another purpose of the present application is to provide a tremella polysaccharide prepared by a eutectic solvent method.

[0010] Still another purpose of the present application is to provide the application of tremella polysaccharide obtained by a eutectic solvent method in preparing drugs for inhibiting the metastasis and / or invasion of breast cancer.

[0011] The above purposes of the present application are achieved by the following technical solutions:

[0012] The present application protects the application of tremella polysaccharide in preparing anti-breast cancer drugs.

[0013] Although a large number of literatures have reported that tremella polysaccharide has various biological activities such as antioxidant, immunomodulatory, and antitumor, but specific to the therapeutic effect and mechanism of a specific disease such as breast cancer, especially the inhibitory effect on the migration and invasion ability of tumor cells, there is still lack of systematic research and clear evidence. However, the present application first discovers that tremella polysaccharide, especially the tremella polysaccharide extracted by a eutectic solvent method, has a significant effect on treating breast cancer, which can inhibit breast cancer through inhibiting cell proliferation, inhibiting cell migration and invasion, etc.

[0014] The present application protects the application of tremella polysaccharide in preparing drugs for inhibiting the proliferation of breast cancer.

[0015] Cell proliferation is the growth, development, reproduction and heredity of cells in the body. The research of the present application shows that tremella polysaccharides prepared by different extraction processes can all inhibit the proliferation of breast cancer cells.

[0016] Further, the tremella polysaccharide includes tremella polysaccharides obtained by a traditional water extraction and alcohol precipitation method and a eutectic solvent method.

[0017] Further, the preparation method of the tremella polysaccharide obtained by the traditional water extraction and alcohol precipitation method comprises the following steps:

[0018] sI. mixing the tremella powder with water, and centrifuging after sufficient extraction at 50-100°C to obtain a tremella polysaccharide extraction solution;

[0019] sII. adding a polar organic solvent to the tremella polysaccharide extraction solution obtained in step sI, mixing, standing, centrifuging, dissolving the precipitate in water, dialyzing with a dialysis bag, collecting the retentate after sufficient dialysis, and freeze-drying the retentate to obtain the tremella polysaccharide.

[0020] Preferably, the polar organic solvent is selected from one or more of ethanol, acetone, isopropanol, and methanol, and more preferably is ethanol. The purpose of alcohol precipitation is to precipitate polysaccharides from the aqueous solution by the dehydrating effect of the polar organic solvent. Considering safety and environmental protection, anhydrous ethanol is usually used as the reagent for alcohol precipitation.

[0021] Further, the mixing ratio of the tremella powder and water is 1:(10-50) g / mL, preferably 1:(15-30) g / mL, and more preferably 1:20 g / mL.

[0022] Further, the volume ratio of the tremella polysaccharide extraction solution and the polar organic solvent is 1:(0.6-20), preferably 1:(2-10), and more preferably 1:4. The addition of the polar organic solvent to the tremella polysaccharide extraction solution results in a concentration of the polar organic solvent in the tremella polysaccharide extraction solution of 37%-95% (referring to the volume concentration of the solvent in the mixture after the addition of the solvent). At this concentration, polysaccharides can be precipitated more fully. Generally, a higher concentration of the solvent will precipitate polysaccharides with smaller molecular weights, but their solubility and other properties will also change. Therefore, the concentration of 37%-95% is commonly used for polysaccharide alcohol precipitation, and the concentration of 60%-90% is preferred, and the concentration of 80% is more preferred. Among them, the concentration of 80% is the most commonly used concentration for alcohol precipitation.

[0023] Preferably, the molecular weight cut-off of the dialysis bag is 8000-14000 Da.

[0024] Preferably, in step sI, the temperature for sufficient extraction is 60-90°C, and more preferably is 80°C.

[0025] Further, the method for preparing the tremella powder comprises the following steps: crushing and sieving the dried tremella, soaking the sieved tremella in a polar organic solvent, filtering, and drying the filter residue to obtain the tremella powder.

[0026] Preferably, the polar organic solvent is selected from one or more of ethanol, acetone, isopropanol, and methanol, and more preferably is ethanol. The main purpose of soaking the tremella powder in the solvent before extracting polysaccharides is to remove fat and small molecular impurities such as pigments, monosaccharides, and low molecular weight saccharides.

[0027] Further, the mass-volume ratio of the screened tremella and the polar organic solvent is 1:(5-30) g / mL, preferably 1:(15-25) g / mL, and more preferably 1:20 g / mL.

[0028] Preferably, the sufficient soaking time is 48-96 h, preferably 60-80 h, and more preferably 72 h.

[0029] Preferably, the sufficient extraction time is 1-4 h, and more preferably 2-3 h.

[0030] Preferably, the sufficient dialysis time is 1-3 days (d).

[0031] Preferably, the screening mesh size is 50-80 mesh, and more preferably 60 mesh.

[0032] Further, the tremella polysaccharide obtained by the low eutectic solvent method is the same as the following tremella polysaccharide preparation.

[0033] The present application also protects a tremella polysaccharide, and the preparation method of the tremella polysaccharide comprises the following steps:

[0034] S1: taking any one or more of urea, citric acid, and glycerol as a hydrogen bond donor, and taking choline chloride as a hydrogen bond acceptor, stirring at 25-100°C until clear and transparent, to obtain a homogeneous low eutectic solvent.

[0035] S2. mixing the tremella powder with the low eutectic solvent obtained in step S1, and performing sufficient extraction at 50-100°C, followed by centrifugation, to obtain a tremella polysaccharide extraction solution;

[0036] S3. adding a polar organic solvent to the tremella polysaccharide extraction solution obtained in step S2, mixing, standing, and centrifuging, dissolving the precipitate in water, and then dialyzing with a dialysis bag, collecting the retentate after sufficient dialysis, and freeze-drying the retentate to obtain a tremella polysaccharide.

[0037] The tremella polysaccharide obtained by the traditional water extraction and alcohol precipitation method has a certain inhibitory effect on the proliferation of breast cancer cells, but it cannot effectively inhibit the migration and invasion of breast cancer cells. The tremella polysaccharide obtained by the low eutectic solvent method not only can inhibit the proliferation of breast cancer cells, but also can significantly inhibit the migration and invasion of breast cancer cells, can effectively block the source of tumor spread, improve the treatment effect and patient survival rate, and reduce the risk of recurrence and metastasis. This multi-target action mode makes the treatment effect more significant and persistent, thereby enhancing the overall treatment effect. The difference in the effects of the above different tremella polysaccharides also indicates that the tremella polysaccharide obtained by the low eutectic solvent method has different bioactive components compared with the tremella polysaccharide obtained by the traditional water extraction and alcohol precipitation method, thereby having significantly different bioactivities.

[0038] Preferably, the hydrogen bond donor is urea.

[0039] Preferably, in step S1, the temperature of the sufficient stirring is 50-90℃, more preferably 80℃.

[0040] Preferably, in step S2, the temperature of the sufficient leaching is 60-90℃, more preferably 80℃.

[0041] Further, the molar ratio of the hydrogen bond donor and the hydrogen bond acceptor is 1:(0.5-4). Specifically, in the present application, the molar ratio of the hydrogen bond donor and the hydrogen bond acceptor is usually 1:1, 1:1.5, 1:2, 1:3, 1:4, 2:1, or an interval range formed by any of the above values.

[0042] Further, the molar ratio of the hydrogen bond donor and the hydrogen bond acceptor is 1:(1-3), more preferably 1:(1.5-2), further more preferably 1:2.

[0043] Further, the mass ratio of the tremella powder and the deep eutectic solvent is 1:(10-50), preferably 1:(15-30), more preferably 1:20.

[0044] Further, the volume ratio of the tremella polysaccharide leaching solution and the polar organic solvent is 1:(0.6-20), preferably 1:(2-10), more preferably 1:4.

[0045] Preferably, the molecular weight cut-off of the dialysis bag is 8000-14000 Da.

[0046] Preferably, the time of the sufficient leaching is 1-4h, more preferably 2-3h.

[0047] Preferably, the time of the sufficient dialysis is 1-3 Day (d).

[0048] Further, the preparation method of the tremella powder comprises the following steps: crushing and sieving the dried tremella, taking the sieved tremella, sufficiently soaking the sieved tremella in a polar organic solvent, filtering, and drying the filter residue to obtain the tremella powder.

[0049] Further, the mass to volume ratio of the sieved tremella and anhydrous ethanol is 1:(5-30) g / mL, preferably 1:(15-25) g / mL, more preferably 1:20 g / mL.

[0050] Preferably, the time of the sufficient soaking is 48-96h, preferably 60-80h, more preferably 72h.

[0051] Further, the total polysaccharide content of the tremella polysaccharide obtained by the deep eutectic solvent method is 68%-79%.

[0052] Further, the protein content of the tremella polysaccharide obtained by the eutectic solvent method is 0.4-0.7%.

[0053] Further, the weight average molecular weight of the tremella polysaccharide obtained by the eutectic solvent method is (1.70-1.75) x 10 5 Da.

[0054] The application also protects the use of the tremella polysaccharide in the preparation of a drug for inhibiting breast cancer metastasis and / or invasion.

[0055] The malignant behavior of a tumor not only includes rapid proliferation, but also includes invasiveness and migration. Even if the direct inhibitory effect of a drug on cell proliferation is not obvious, if it significantly inhibits the migration and invasion ability of cells, it can play an important role in inhibiting tumor spread and metastasis. The migration and invasion of tumor cells are the main processes of tumor spread and metastasis, and preventing the migration and invasion of tumor cells can directly block the further spread of tumors in the body, thereby reducing the burden of tumors and damage to other organs. Although proliferation is the basis of tumor growth, even if tumor cell proliferation is inhibited, if the migration and invasion ability of the tumor cells is not limited, the tumor can still form new lesions by transferring to other sites. Therefore, in some cases, it is more important to inhibit the migration and invasion ability of tumor cells than to inhibit proliferation, especially in preventing tumor metastasis.

[0056] Although cell proliferation, migration and invasion are all key links in the process of tumor growth and metastasis, they are regulated by different signal pathways. The tremella polysaccharide obtained by the traditional water extraction and alcohol precipitation method has a certain inhibitory effect on the proliferation of breast cancer cells, but it cannot effectively inhibit the migration and invasion of breast cancer cells. The tremella polysaccharide obtained by the eutectic solvent method not only can inhibit the proliferation of breast cancer cells, but also can significantly inhibit the migration and invasion of breast cancer cells, can effectively block the source of tumor spread, improve the treatment effect and patient survival rate, and reduce the risk of recurrence and metastasis. This multi-target action mode makes the treatment effect more significant and persistent, thereby enhancing the overall effect of treatment.

[0057] Compared with the prior art, the present application has the following beneficial effects: the present application first discovers that tremella polysaccharide has an anti-breast cancer effect, which can inhibit breast cancer through the pathways of inhibiting cell proliferation, inhibiting cell migration and invasion, etc. Compared with the tremella polysaccharide obtained by the traditional water extraction and alcohol precipitation method, the tremella polysaccharide obtained by the eutectic solvent method has lower total sugar content and average molecular weight, higher protein content, and not only can inhibit the proliferation of breast cancer cells, but also can significantly inhibit the migration and invasion of breast cancer cells. This multi-target action mode can effectively block the source of tumor spread, improve the treatment effect and patient survival rate, reduce the risk of recurrence and metastasis, enhance the overall effect of treatment, and has good application prospects in the treatment of breast cancer. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 Figure 4 is a data statistical graph of the effect of tremella polysaccharide on the proliferation of MDA-MB-231 breast cancer cells; the marked * indicates a significant difference compared with the control group, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, ns indicates no significant effect; a indicates TFP-H, b indicates TFP-G, c indicates TFP-C, d indicates TFP-U, Control indicates the blank control group.

[0059] Figure 2 Figure 5 is a data statistical graph of the effect of tremella polysaccharide on the migration of MDA-MB-231 breast cancer cells; the marked * indicates a significant difference compared with the control group, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, ns indicates no significant effect; a indicates TFP-H, b indicates TFP-G, c indicates TFP-C, d indicates TFP-U, Control indicates the blank control group.

[0060] Figure 3 Figure 6 is a data statistical graph of the effect of tremella polysaccharide on the invasion of MDA-MB-231 breast cancer cells; the marked * indicates a significant difference compared with the control group, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, ns indicates no significant effect; a indicates TFP-H, b indicates TFP-G, c indicates TFP-C, d indicates TFP-U, Control indicates the blank control group. DETAILED DESCRIPTION

[0061] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0062] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0063] Example 1 Tremella polysaccharide extracted by low eutectic solvent (urea + choline chloride)

[0064] The preparation method of tremella polysaccharide extracted by low eutectic solvent (urea + choline chloride) comprises the following steps:

[0065] S1. Dry the dried tremella in a pulverizer, pass through a 60-mesh sieve, soak in anhydrous ethanol at a ratio of 1:20 (g / mL) for 72 h to remove lipids and small molecule impurities, suction filter, and dry the filter residue at 60°C to obtain tremella powder, which is sealed and stored for use.

[0066] S2. Urea was selected as hydrogen bond donor and choline chloride as hydrogen bond acceptor to prepare clear and homogeneous DES at 80℃ with a molar ratio of 1:2. The tremella powder obtained in step S1 was mixed with the DES at a ratio of 1:20 (w / w) and extracted at 80℃ for 140 min. The solution was centrifuged (10000 rpm, 10 min) to obtain a tremella polysaccharide solution.

[0067] S3. The tremella polysaccharide solution obtained in step S2 was added to 4 times the volume of anhydrous ethanol and alcohol precipitated at 4℃ overnight. The precipitate was centrifuged (10000 rpm, 5 min) and then redissolved in deionized water. After dialysis for three days in a dialysis bag with a molecular weight of 8000-14000 Da, the precipitate was freeze-dried to obtain a tremella polysaccharide, which was named TFP-U.

[0068] Example 2 Tremella polysaccharide extracted by deep eutectic solvent (citric acid + choline chloride)

[0069] The preparation method of the tremella polysaccharide extracted by the deep eutectic solvent (citric acid + choline chloride) comprises the following steps:

[0070] S1. Dry tremella was crushed in a crusher and sieved through a 60-mesh sieve. The tremella was soaked in anhydrous ethanol at a ratio of 1:20 (g / mL) for 72 h to remove lipids and small molecule impurities. The filtrate was filtered and the residue was dried at 60℃. The tremella powder was sealed and stored for later use.

[0071] S2. Citric acid was selected as the hydrogen bond donor and choline chloride as the hydrogen bond acceptor to prepare a clear and homogeneous DES at 80℃ with a molar ratio of 1:2. The tremella powder obtained in step S1 was mixed with the DES at a ratio of 1:20 (w / w) and extracted at 80℃ for 140 min. The solution was centrifuged (10000 rpm, 10 min) to obtain a tremella polysaccharide solution.

[0072] S3. The tremella polysaccharide solution obtained in step S2 was added to 4 times the volume of anhydrous ethanol and alcohol precipitated at 4℃ overnight. The precipitate was centrifuged (10000 rpm, 5 min) and then redissolved in deionized water. After dialysis for three days in a dialysis bag with a molecular weight of 8000-14000 Da, the precipitate was freeze-dried to obtain a tremella polysaccharide, which was named TFP-C.

[0073] Example 3 Tremella polysaccharide extracted by deep eutectic solvent (glycerol + choline chloride)

[0074] The preparation method of the tremella polysaccharide extracted by the deep eutectic solvent (glycerol + choline chloride) comprises the following steps:

[0075] S1. The dried Tremella fuciformis was crushed in a pulverizer and passed through a 60-mesh sieve. The crushed Tremella fuciformis was soaked in anhydrous ethanol at a ratio of 1:20 (g / mL) for 72 h to remove lipids and small molecular impurities. The filtrate was extracted and the residue was dried at 60°C to obtain Tremella fuciformis powder, which was stored in a sealed container.

[0076] S2. Glycerol was selected as the hydrogen bond donor and choline chloride as the hydrogen bond acceptor to prepare a clear and uniform DES at 80°C. The Tremella fuciformis powder obtained in step S1 was mixed with the DES at a ratio of 1:20 (w / w) and extracted at 80°C for 140 min. The Tremella fuciformis polysaccharide solution was obtained by centrifugation (10000 rpm, 10 min).

[0077] S3. The Tremella fuciformis polysaccharide solution obtained in step S2 was added to 4 times the volume of anhydrous ethanol and alcohol precipitated at 4°C overnight. The precipitate was resuspended in deionized water and dialyzed in a dialysis bag with a molecular weight cut-off of 8000-14000 Da for three days. The freeze-dried Tremella fuciformis polysaccharide obtained was named TFP-G.

[0078] The method for preparing Tremella fuciformis polysaccharide by water extraction comprises the following steps:

[0079] S1. The dried Tremella fuciformis was crushed in a pulverizer and passed through a 60-mesh sieve. The crushed Tremella fuciformis was soaked in anhydrous ethanol at a ratio of 1:20 (g / mL) for 72 h to remove lipids and small molecular impurities. The filtrate was extracted and the residue was dried at 60°C to obtain Tremella fuciformis powder, which was stored in a sealed container.

[0080] S2. The Tremella fuciformis powder obtained in step S1 was mixed with hot water at a ratio of 1:20 (w / w) and extracted at 80°C for 140 min. The water-extracted Tremella fuciformis polysaccharide solution was obtained by centrifugation (10000 rpm, 10 min).

[0081] S2. The water-extracted Tremella fuciformis polysaccharide solution obtained in step S2 was added to 4 times the volume of anhydrous ethanol and alcohol precipitated at 4°C overnight. The precipitate was resuspended in deionized water and dialyzed in a dialysis bag with a molecular weight cut-off of 8000-14000 Da for three days. The freeze-dried Tremella fuciformis polysaccharide obtained was named TFP-H.

[0082] 1. Experimental method

[0083] The total sugar content, uronic acid content, and protein content of the Tremella fuciformis polysaccharide were determined by the phenol-sulfuric acid method, the m-hydroxybenzene method, and the Coomassie brilliant blue method, respectively. In addition, the monosaccharide composition of the Tremella fuciformis polysaccharide obtained in Examples 1-3 and Comparative Example 1 was determined by high-performance liquid chromatography with PMP (1-phenyl-3-methyl-5-pyrazolone) derivatization.

[0084] 2. Experimental results

[0085] Table 1 Yield and chemical composition of tremella polysaccharides obtained by different extraction processes

[0086]

[0087]

[0088] The yield and chemical composition of tremella polysaccharides obtained by different extraction processes are shown in Table 1. Compared with the tremella polysaccharides obtained by traditional water extraction and alcohol precipitation, the total sugar content, uronic acid content (except for TFP-C obtained in Example 2) and weight average molecular weight of the tremella polysaccharides obtained by the eutectic solvent method are lower, and the protein content is slightly higher. Further monosaccharide composition analysis of the polysaccharides obtained in the examples and comparative examples found that the tremella polysaccharides were mainly composed of mannose, glucuronic acid, glucose, xylose and fucose. Among them, the mannose content was the highest in TFP-C (46.65%) > TFP-U (43.03%) > TFP-H (39.42%) > TFP-G (31.73%), followed by glucose TFP-G (42.05%) > TFP-H (30.71%) > TFP-C (28.23%) > TFP-U (22.27%). The above differences in chemical composition endow the tremella polysaccharides with different biological activities.

[0089] Experimental Example 2 Anti-breast cancer experiment

[0090] 1. Experimental method

[0091] The TFPs (TFP-U, TFP-C, TFP-G, TFP-H) obtained in Examples 1-3 and Comparative Example 1 were subjected to MTT cytotoxicity test, wound healing experiment and cell migration experiment, respectively, to evaluate the effects of tremella polysaccharides obtained by different extraction methods on the functions of breast cancer cells.

[0092] (1) MTT cytotoxicity test

[0093] The control group (control) was a p H 7.4 PBS solution. The administration group was intragastrically administered with different concentrations of TFP, with a concentration range of 10-640 μg / mL. Incubation was performed in a 37°C, 5% CO2 incubator for 48 h, 5 mg / mL MTT was added, and incubation was performed in a dark incubator for 4 h. After 4 h of incubation, dimethyl sulfoxide (DMSO) solution was added, and the OD value of each well was measured at 490 nm. After 4 h of incubation, DMSO was added at 490 nm, and the OD value of each well was measured again.

[0094] (2) Wound healing experiment

[0095] MDA-MB-231 cells were vertically cultured in a six-well plate, and the control group was added with DMEM medium containing 2% fetal bovine serum (FBS), and the drug group was added with TFP (306.8 μg / mL) obtained by different extraction processes. The wound healing rate was calculated by ImageJ 1.53K.

[0096] (3) Cell migration test

[0097] MDA-MB-231 cell suspension and 306.8 μg / mL tremella polysaccharide treatment medium or blank medium (control group) were added to the upper chamber, and complete medium containing 20% FBS was added to the lower chamber. After 48 h of culture, 4% paraformaldehyde was used for fixation for 30 min, PBS was used for washing, and 0.1% crystal violet solution was used for staining for 30 min. Five fields were randomly selected under a microscope for photography and counting.

[0098] 2. Experimental results

[0099] (1) Effect of tremella polysaccharide on breast cancer cell proliferation

[0100] MTT method was used to observe the effect of tremella polysaccharide on the growth of breast cancer cells MDA-MB-231. The inhibitory effect of tremella polysaccharide extracted by four different extraction methods on breast cancer cell proliferation showed time and concentration dependence. Among them, the inhibitory effect of the four different extraction methods on tremella polysaccharide was: TFP-U > TFP-C > TFP-G > TFP-H. Among them, TFP-U had the most significant inhibitory effect, with an IC 50 value of 306.8 μg / mL( Figure 1 ).

[0101] (2) Effect of tremella polysaccharide on breast cancer cell migration and invasion

[0102] The wound healing experiment results showed that the MDA-MB-231 cells in the control group gradually healed after 48 h of culture. Compared with the control group, the cell migration rate of the tremella polysaccharide treatment group was reduced, but the tremella polysaccharide TFP-H obtained by the comparison 1 had no significant difference compared with the control group, while the tremella polysaccharide extracted by the eutectic solvent had significant difference compared with the control group. The inhibitory effect of migration was TFP-U > TFP-C > TFP-G > TFP-H > control group( Figure 2 ). Among them, the tremella polysaccharide TFP-U obtained by urea + choline chloride in the eutectic solvent had the best inhibitory effect on migration, and the tremella polysaccharide TFP-H obtained by water extraction had significantly lower inhibitory effect on migration than other eutectic solvent groups.

[0103] The results of the migration and invasion experiment show that the number of invaded cells in the tremella polysaccharide treatment groups is lower than that in the control group, but the number of invaded cells in the silver ear polysaccharide TFP-H obtained by the comparative example 1 has no significant difference compared with the control group, and the number of invaded cells in the silver ear polysaccharide extracted by the eutectic solvent has significant difference compared with the control group. The number of invaded cells in the silver ear polysaccharide TFP-U is significantly lower than that in other groups. It can be seen that the inhibition of invasion between groups is: the inhibition of breast cancer cell proliferation, migration and invasion TFP-U>TFP-C>TFP-G>TFP-H>control group. These results show that the silver ear polysaccharide extracted by the eutectic solvent has a significant anti-breast cancer effect, and the TFP-U has a better anti-breast cancer effect. Figure 3

[0104] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.​

Claims

1. The use of Tremella polysaccharide in the preparation of anti-breast cancer drugs, characterized in that, The preparation method of the tremella polysaccharide comprises the following steps: S1: taking any one or more of urea, citric acid and glycerol as a hydrogen bond donor, and taking choline chloride as a hydrogen bond acceptor, stirring at 25-100 DEG C until clear and transparent to obtain a homogeneous eutectic solvent; S2. The tremella powder is mixed with the eutectic solvent obtained in step S1, and after sufficient extraction at 50-100 DEG C, centrifugation is performed to obtain a tremella polysaccharide extraction solution; S3. A polar organic solvent is added to the tremella polysaccharide extraction solution obtained in step S2, and after mixing, standing, centrifugation, the precipitate is dissolved in water, and then dialysis is performed using a dialysis bag, and after sufficient dialysis, the retention liquid is collected, and the retention liquid is freeze-dried to obtain the tremella polysaccharide. The polar organic solvent is selected from one or more of ethanol, acetone, isopropyl alcohol and methanol.

2. Use according to claim 1, characterized in that, The anti-breast cancer is to inhibit the proliferation of breast cancer cells.

3. Use according to claim 1, characterized in that, The anti-breast cancer is to inhibit the metastasis and / or invasion of breast cancer cells.

4. The use according to claim 1, characterized in that, The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1: (0.5-4).

5. The use according to claim 1, characterized in that, The mass ratio of the tremella powder to the eutectic solvent is 1: (10-50).

6. The use according to claim 1, characterized in that, The volume ratio of the tremella polysaccharide extraction solution to the polar organic solvent is 1: (0.6-20).

7. Use according to claim 1, characterized in that, The preparation method of the tremella powder comprises the following steps: crushing and sieving the dried tremella, and then soaking the sieved tremella in a polar organic solvent, filtering, and drying the filter residue to obtain the tremella powder.

8. Use according to claim 7, characterized in that, The mass-to-volume ratio of the sieved tremella to the polar organic solvent is 1g: (5-30)mL.

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