Tremella polysaccharide prepared by deep eutectic solvent method and application of tremella polysaccharide in preparation of anti-breast cancer drugs

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to tremella polysaccharide prepared by a deep eutectic solvent method and application of the tremella polysaccharide in preparation of anti-breast cancer medicines. The tremella polysaccharide extracted by the eutectic solvent has an obvious effect of treating the breast cancer for the first time, and can resist the breast cancer by inhibiting cell proliferation, cell migration and invasion and the like. Compared with tremella polysaccharide obtained through a traditional water extraction and alcohol precipitation method, the tremella polysaccharide obtained through the deep eutectic solvent method is low in total sugar content and weight-average molecular weight and slightly high in protein content, inhibition of proliferation of breast cancer cells can be remarkably improved, migration and invasion of the breast cancer cells can be remarkably inhibited, and the tremella polysaccharide can be used for treating breast cancer. The multi-target action mode can effectively block the source of tumor diffusion, improve the treatment effect and the survival rate of patients, reduce the recurrence and metastasis risks and enhance the overall treatment effect, and has a good application prospect in treatment of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology. More specifically, it relates to Tremella fuciformis polysaccharides prepared by the deep eutectic solvent method and their application in the preparation of anti-breast cancer drugs. Background Art

[0002] In recent years, with the continuous in-depth research of biotechnology and natural products, scientists have gradually turned their attention to the exploration and application of traditional Chinese medicinal materials and their active ingredients. Tremella fuciformis, as a fungal food material with a long history and both medicinal and edible properties, has attracted much attention due to its rich nutritional components and potential medicinal value. Tremella fuciformis polysaccharides (TFPs), as one of the main active ingredients in Tremella fuciformis, have shown broad application prospects in the fields of medicine, health care, and functional foods due to their unique biological activities and good biocompatibility.

[0003] Deep Eutectic Solvents (DESs), as a new type of green solvent system, have attracted extensive attention in the field of natural product extraction in recent years due to their simple preparation, low cost, biodegradability, and good solubility for various substances. Compared with traditional organic solvents, deep eutectic solvents can not only effectively retain the structure and biological activities of 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 using deep eutectic solvents to extract active ingredients from various plants and microorganisms, including polysaccharide compounds. For example, Chinese patent application CN113354754A discloses a method for extracting Tremella fuciformis polysaccharides from Tremella fuciformis spore fermentation broth using deep eutectic solvents. By optimizing the solvent composition and extraction conditions, the extraction efficiency and purity of Tremella fuciformis polysaccharides are effectively improved. However, this patent only focuses on the extraction process itself and does not deeply explore or disclose the specific biological activities and potential applications of the extracted Tremella fuciformis polysaccharides, especially their potential value in the field of medical treatment.

[0005] In addition, although a large number of literatures have reported that Tremella fuciformis 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 the inhibitory effect on its migration and invasion ability. Moreover, there are few reports that deep eutectic solvent extraction can significantly improve the anti-tumor activity of Tremella fuciformis polysaccharides. This research gap not only restricts the further expansion of Tremella fuciformis polysaccharides in clinical applications but also provides room for exploration and innovation for scientific researchers.

[0006] In summary, although certain progress has been made in the extraction methods of tremella polysaccharides and their general biological activities in the prior art, there is still a lack of research in the treatment effects on specific diseases. Therefore, developing new extraction technologies and deeply exploring the potential of tremella polysaccharides in disease treatment, especially their inhibitory effects on specific tumors (such as breast cancer), has important scientific significance and clinical application value. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art that focuses on the extraction process itself and does not deeply explore or disclose the specific biological activities and potential applications of the extracted tremella polysaccharides, and to provide the application of tremella polysaccharides in the preparation of anti-breast cancer drugs.

[0008] The object of the present invention is to provide the application of tremella polysaccharides in the preparation of drugs for inhibiting the proliferation of breast cancer.

[0009] Another object of the present invention is to provide a tremella polysaccharide prepared by the deep eutectic solvent method.

[0010] Another object of the present invention is to provide the application of the tremella polysaccharide obtained by the deep eutectic solvent method in the preparation of drugs for inhibiting breast cancer metastasis and / or invasion.

[0011] The above objects of the present invention are achieved by the following technical solutions:

[0012] The present invention protects the application of tremella polysaccharides in the preparation of anti-breast cancer drugs.

[0013] Although a large number of existing literatures report that tremella polysaccharides have various biological activities such as antioxidant, immunomodulatory, and anti-tumor, specific to the treatment effects and mechanisms of a certain specific disease such as breast cancer, especially the inhibitory effects on the migration and invasion abilities of tumor cells, there is still a lack of systematic research and clear evidence. The present invention for the first time discovers that tremella polysaccharides, especially tremella polysaccharides extracted by deep eutectic solvents, have obvious effects in treating breast cancer and can fight breast cancer by inhibiting cell proliferation, cell migration and invasion, etc.

[0014] The present invention protects the application of tremella polysaccharides in the preparation of drugs for inhibiting the proliferation of breast cancer.

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

[0016] Furthermore, the tremella polysaccharides include tremella polysaccharides obtained by the traditional water extraction and alcohol precipitation method and the deep eutectic solvent method.

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

[0018] sI. Mix tremella powder with water, fully extract at 50 - 100 °C and then centrifuge to obtain the tremella polysaccharide extract.

[0019] sII. Add a polar organic solvent to the tremella polysaccharide extract obtained in step sI, mix well, let stand, centrifuge, dissolve the precipitate in water and then dialyze using a dialysis bag. After sufficient dialysis, collect the retention solution and lyophilize the retention solution to obtain tremella polysaccharide.

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

[0021] Furthermore, 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] Furthermore, the volume ratio of the tremella polysaccharide extract to the polar organic solvent is 1:(0.6 - 20), preferably 1:(2 - 10), and more preferably 1:4. Adding a polar organic solvent to the tremella polysaccharide extract makes the concentration of the polar organic solvent in the tremella polysaccharide extract 37% - 95% (referring to the volume concentration of the solvent in the mixture after adding the solvent). At this concentration, polysaccharides can be precipitated more fully. Generally, a higher concentration of the solvent will precipitate polysaccharides with a smaller molecular weight, but their solubility and other properties will also change. Therefore, the concentration of 37% - 95% is commonly used for polysaccharide alcohol precipitation, preferably the concentration of 60% - 90%, and more preferably 80% concentration. Among them, 80% concentration is the most commonly used alcohol precipitation concentration.

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

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

[0025] Furthermore, the preparation method of the tremella powder includes the following steps: crush and sieve the dried tremella, soak the sieved tremella fully in a polar organic solvent, filter, and dry 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 ethanol. The main purpose of soaking the tremella powder in a solvent before extracting polysaccharides is to degrease and remove small molecule impurities such as pigments, monosaccharides, and low molecular weight saccharides.

[0027] Further, the mass-volume ratio of the sieved 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 time for sufficient soaking is 48-96 h, preferably 60-80 h, and more preferably 72 h.

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

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

[0031] Preferably, the mesh number for sieving is 50-80 meshes, and preferably 60 meshes.

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

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

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

[0035] S2. Mix the tremella powder with the deep eutectic solvent obtained in step S1, fully extract at 50-100 °C and then centrifuge to obtain a tremella polysaccharide extract;

[0036] S3. Add a polar organic solvent to the tremella polysaccharide extract obtained in step S2, mix well, let stand, and centrifuge. Take the precipitate, dissolve it in water, then dialyze it using a dialysis bag. After sufficient dialysis, collect the retention solution, and lyophilize the retention solution to obtain the tremella polysaccharide.

[0037] The tremella polysaccharide obtained by the traditional water extraction and alcohol precipitation method mentioned above 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. However, the tremella polysaccharide obtained by the deep eutectic solvent method can not only inhibit the proliferation of breast cancer cells, but also significantly inhibit the migration and invasion of breast cancer cells, can effectively block the source of tumor spread, improve the treatment effect and the survival rate of patients, reduce the risk of recurrence and metastasis. This multi-target action mode makes the treatment effect more significant and lasting, thus enhancing the overall treatment effect. The difference in the effects of the above different tremella polysaccharides also shows that the tremella polysaccharide obtained by the deep eutectic solvent method has different bioactive components compared with the tremella polysaccharide obtained by the traditional water extraction and alcohol precipitation method, and thus has significantly different biological activities.

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

[0039] Preferably, in step S1, the temperature for the sufficient stirring is 50 to 90 °C, more preferably 80 °C.

[0040] Preferably, in step S2, the temperature for the sufficient extraction is 60 to 90 °C, more preferably 80 °C.

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

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

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

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

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

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

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

[0048] Furthermore, the method for preparing the tremella powder includes the following steps: pulverize and sieve the dried tremella, take the sieved tremella and soak it sufficiently in a polar organic solvent, filter, and take the filter residue and dry it to obtain the tremella powder.

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

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

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

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

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

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

[0055] The malignant behavior of tumors not only includes rapid proliferation, but also includes invasiveness and migratoriness. 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. Preventing the migration and invasion of tumor cells can directly block the further spread of tumors in the body, thereby reducing the tumor burden and damage to other organs. Although proliferation is the basis of tumor growth, even if tumor cell proliferation is inhibited, if its migration and invasion abilities are not restricted, the tumor may still form new lesions by metastasizing to other sites. Therefore, in some cases, inhibiting the migration and invasion ability of tumor cells is more important than inhibiting 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 signaling pathways. The tremella polysaccharide obtained by the traditional water extraction and alcohol precipitation method mentioned above 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 deep eutectic solvent method can not only inhibit the proliferation of breast cancer cells, but also significantly inhibit the migration and invasion of breast cancer cells, which can effectively block the source of tumor spread, improve the treatment effect and patient survival rate, reduce the risk of recurrence and metastasis. This multi-target action mode makes the treatment effect more significant and lasting, thus enhancing the overall treatment effect.

[0057] Compared with the prior art, the present invention has the following beneficial effects: The present invention discovers for the first time that tremella polysaccharide has an anti-breast cancer effect and can resist breast cancer through pathways such as inhibiting cell proliferation, inhibiting cell migration and invasion. Compared with the tremella polysaccharide obtained by the traditional water extraction and alcohol precipitation method, the total sugar content and average molecular weight of the tremella polysaccharide obtained by the deep eutectic solvent method are lower, the protein content is higher, and it can not only inhibit the proliferation of breast cancer cells, but also 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, and enhance the overall treatment effect, having good application prospects in the treatment of breast cancer. Brief Description of the Drawings

[0058] Figure 1 It is a statistical chart of the data on the effect of Tremella polysaccharide on the proliferation of MDA-MB-231 breast cancer cells; the annotation * 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 represents TFP-H, b represents TFP-G, c represents TFP-C, d represents TFP-U, and Control represents the blank control group.

[0059] Figure 2 It is a statistical chart of the data on the effect of Tremella polysaccharide on the migration of MDA-MB-231 breast cancer cells; the annotation * 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 represents TFP-H, b represents TFP-G, c represents TFP-C, d represents TFP-U, and Control represents the blank control group.

[0060] Figure 3 It is a statistical chart of the data on the effect of Tremella polysaccharide on the invasion of MDA-MB-231 breast cancer cells; the annotation * 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 represents TFP-H, b represents TFP-G, c represents TFP-C, d represents TFP-U, and Control represents the blank control group. Detailed Embodiments

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

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

[0063] Example 1 Tremella polysaccharide extracted using a deep eutectic solvent (urea + choline chloride)

[0064] The preparation method of Tremella polysaccharide extracted using a deep eutectic solvent (urea + choline chloride) includes the following steps:

[0065] S1. Crush the dried Tremella in a pulverizer, pass through a 60-mesh sieve, soak it in absolute ethanol at a ratio of 1:20 (g / mL) for 72 h to remove lipids and small molecule impurities, filter by suction, dry the filter residue at 60 °C to obtain Tremella powder, and seal and store it for later use.

[0066] S2. Select urea as the hydrogen bond donor and choline chloride as the hydrogen bond acceptor. Prepare a clear and homogeneous DES at a molar ratio of 1:2 at 80 °C. Mix the tremella powder obtained in step S1 with DES at a ratio of 1:20 (w / w), extract at 80 °C for 140 min, and centrifuge (10000 rpm, 10 min) to obtain a tremella polysaccharide solution.

[0067] S3. Add 4 volumes of absolute ethanol to the tremella polysaccharide solution obtained in step S2 above, perform alcohol precipitation overnight at 4 °C, centrifuge (10000 rpm, 5 min), then redissolve the precipitate in deionized water, dialyze using an 8000 - 14000 Da dialysis bag for three days, and then freeze-dry. The obtained tremella polysaccharide is named TFP-U.

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

[0069] A preparation method of tremella polysaccharide extracted using a deep eutectic solvent (citric acid + choline chloride) includes the following steps:

[0070] S1. Crush the dried tremella in a crusher, pass through a 60-mesh sieve, soak in absolute ethanol at a ratio of 1:20 (g / mL) for 72 h to remove lipids and small molecule impurities, perform suction filtration, and dry the filter residue at 60 °C to obtain tremella powder, which is sealed and stored for later use.

[0071] S2. Select citric acid as the hydrogen bond donor and choline chloride as the hydrogen bond acceptor. Prepare a clear and homogeneous DES at a molar ratio of 1:2 at 80 °C. Mix the tremella powder obtained in step S1 with DES at a ratio of 1:20 (w / w), extract at 80 °C for 140 min, and centrifuge (10000 rpm, 10 min) to obtain a tremella polysaccharide solution.

[0072] S3. Add 4 volumes of absolute ethanol to the tremella polysaccharide solution obtained in step S2 above, perform alcohol precipitation overnight at 4 °C, centrifuge (10000 rpm, 5 min), then redissolve the precipitate in deionized water, dialyze using an 8000 - 14000 Da dialysis bag for three days, and then freeze-dry. The obtained tremella polysaccharide is named TFP-C.

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

[0074] A preparation method of tremella polysaccharide extracted using a deep eutectic solvent (glycerol + choline chloride) includes the following steps:

[0075] S1. Crush the dried tremella fuciformis in a pulverizer, sieve it through a 60-mesh sieve, soak it in absolute ethanol at a ratio of 1:20 (g / mL) for 72 h to remove lipids and small molecule impurities, filter by suction, dry the filter residue at 60 °C to obtain tremella fuciformis powder, and store it sealed for later use.

[0076] S2. Select glycerol as the hydrogen bond donor and choline chloride as the hydrogen bond acceptor, and prepare a clear and homogeneous DES at a molar ratio of 1:2 at 80 °C. Mix the tremella fuciformis powder obtained in step S1 with DES at a ratio of 1:20 (w / w), extract at 80 °C for 140 min, and centrifuge (10000 rpm, 10 min) to obtain a tremella fuciformis polysaccharide solution.

[0077] S3. Add 4 volumes of absolute ethanol to the tremella fuciformis polysaccharide solution obtained in step S2 above, perform alcohol precipitation overnight at 4 °C, centrifuge (10000 rpm, 5 min), then redissolve the precipitate in deionized water, dialyze with an 8000 - 14000 Da dialysis bag for three days and then freeze-dry. The obtained tremella fuciformis polysaccharide is named TFP-G. Tremella fuciformis polysaccharide prepared by the traditional extraction method in Comparative Example 1

[0078] The preparation method of tremella fuciformis polysaccharide extracted with water includes the following steps:

[0079] S1. Crush the dried tremella fuciformis in a pulverizer, sieve it through a 60-mesh sieve, soak it in absolute ethanol at a ratio of 1:20 (g / mL) for 72 h to remove lipids and small molecule impurities, filter by suction, dry the filter residue at 60 °C to obtain tremella fuciformis powder, and store it sealed for later use.

[0080] S2. Mix the tremella fuciformis powder obtained in step S1 with hot water at a ratio of 1:20 (w / w), extract at 80 °C for 140 min, and centrifuge (10000 rpm, 10 min) to obtain a water-extracted tremella fuciformis polysaccharide solution.

[0081] S2. Add 4 volumes of absolute ethanol to the water-extracted tremella fuciformis polysaccharide solution obtained in step S2 above, perform alcohol precipitation overnight at 4 °C, centrifuge (10000 rpm, 5 min), then redissolve the precipitate in deionized water, dialyze with an 8000 - 14000 Da dialysis bag for three days and then freeze-dry. The obtained tremella fuciformis polysaccharide is named TFP-H. Determination of the chemical properties of tremella fuciformis polysaccharide in the experimental example

[0082] 1. Experimental method

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

[0084] 2. Experimental Results

[0085] Table 1 Yields and Chemical Compositions of Tremella fuciformis Polysaccharides Obtained by Different Extraction Processes

[0086]

[0087]

[0088] The yields and chemical compositions of Tremella fuciformis polysaccharides obtained by different extraction processes are shown in Table 1. Compared with the Tremella fuciformis polysaccharides obtained by traditional water extraction and ethanol precipitation, the total sugar content, uronic acid content (except for TFP-C obtained in Example 2), and weight-average molecular weight of the Tremella fuciformis polysaccharides extracted by the deep 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 Tremella fuciformis polysaccharides are mainly composed of mannose, glucuronic acid, glucose, xylose, and fucose. Among them, the mannose content is the highest, 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%). These differences in chemical composition endow Tremella fuciformis 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 respectively subjected to MTT cytotoxicity test, wound healing experiment, and cell migration experiment to evaluate the effects of Tremella fuciformis polysaccharides obtained by different extraction methods on the functions of breast cancer cells.

[0092] (1) MTT Cytotoxicity Test

[0093] The control group was the pH 7.4 PBS solution. The administration group was gavaged with different concentrations of TFP, and the concentration range was 10-640 μg / mL. Incubate in a 37°C, 5% CO 2 incubator for 48 h, add 5 mg / mL MTT, and incubate in the dark incubator for 4 h. After incubating for 4 h, add dimethyl sulfoxide (DMSO) solution, and measure the OD value of each well at 490 nm. After incubating for 4 h, add DMSO at 490 nm, and measure the OD value of each well again.

[0094] (2) Wound Healing Experiment

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

[0096] (3) Cell migration assay

[0097] An MDA-MB-231 cell suspension and Tremella fuciformis polysaccharide-treated medium or blank medium (control group) at 306.8 μg / mL were added to the upper chamber, and a complete medium containing 20% FBS was added to the lower chamber. After culturing for 48 h, it was fixed with 4% paraformaldehyde for 30 min, washed with PBS, and stained with 0.1% crystal violet solution for 30 min. Five visual fields were randomly selected for photographing and counting under the microscope.

[0098] 2. Experimental results

[0099] (1) Effect of Tremella fuciformis polysaccharide on the proliferation of breast cancer cells

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

[0101] (2) Effect of Tremella fuciformis polysaccharide on the migration and invasion of breast cancer cells

[0102] The results of the wound healing experiment 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 in the Tremella fuciformis polysaccharide treatment group decreased. However, there was no significant difference between the Tremella fuciformis polysaccharide TFP-H obtained in Comparative Example 1 and the control group, while the Tremella fuciformis polysaccharide extracted using a deep eutectic solvent had significant differences compared with the control group. The inhibitory effect on migration was TFP-U > TFP-C > TFP-G > TFP-H > control group ( Figure 2 ). Among them, the Tremella fuciformis polysaccharide TFP-U obtained from urea + choline chloride in the deep eutectic solvent had the best inhibitory effect on migration, while the inhibitory effect of the Tremella fuciformis polysaccharide TFP-H obtained by water extraction was significantly lower than that of other deep eutectic solvent groups.

[0103] The results of the transwell invasion assay showed that the number of invasive cells in the tremella polysaccharide treatment groups was lower than that in the control group. However, there was no significant difference between the tremella polysaccharide TFP-H obtained in Example 1 and the control group, while the tremella polysaccharides extracted using deep eutectic solvents showed significant differences compared with the control group. Among them, the number of invasive cells of tremella polysaccharide TFP-U was significantly lower than that of other groups. It can be seen that the inhibitory effects on invasion among the groups were as follows: the inhibitory effects on the proliferation, migration and invasion of breast cancer cells were TFP-U > TFP-C > TFP-G > TFP-H > control group. These results indicate that the tremella polysaccharides extracted using deep eutectic solvents have significant anti-breast cancer effects, and among them, TFP-U has a better anti-breast cancer effect( Figure 3 ).

[0104] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of Tremella polysaccharide in the preparation of anti-breast cancer drugs.

2. Application of Tremella polysaccharide in the preparation of drugs for inhibiting the proliferation of breast cancer.

3. A Tremella polysaccharide, characterized in that The preparation method of Tremella polysaccharide comprises the following steps: S1: using any one or more of urea, citric acid, and glycerol as hydrogen bond donors and choline chloride as hydrogen bond acceptor, stirring at 25-100° C. until clear and transparent to obtain a uniform low eutectic solvent; S2. Mixing the Tremella powder with the low eutectic solvent obtained in step S1, fully extracting at 50-100° C. and centrifuging to obtain a Tremella polysaccharide extract; S2. Add a polar organic solvent to the Tremella polysaccharide extract obtained in step S2, mix, let stand, and centrifuge. Dissolve the precipitate in water and dialyze it using a dialysis bag. After sufficient dialysis, collect the retained solution, and lyophilize the retained solution to obtain Tremella polysaccharide.

4. The Tremella polysaccharide according to claim 3, characterized in that The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:(0.5-4).

5. The Tremella polysaccharide according to claim 3, characterized in that The mass ratio of the tremella powder to the low eutectic solvent is 1:(10-50).

6. The Tremella polysaccharide according to claim 3, characterized in that The volume ratio of the Tremella polysaccharide extract to the polar organic solvent is 1:(0.6-20).

7. The Tremella polysaccharide according to claim 3, characterized in that The preparation method of the tremella powder comprises the following steps: crushing and sieving the dried tremella, fully soaking the sieved tremella in a polar organic solvent, filtering, and drying the filter residue to obtain the tremella powder.

8. The Tremella polysaccharide according to any one of claims 3 to 7, characterized in that The polar organic solvent is selected from one or more of ethanol, acetone, isopropanol and methanol.

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

10. Use of the Tremella polysaccharide according to any one of claims 3 to 9 in the preparation of a medicament for inhibiting breast cancer metastasis and / or invasion.

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