Preparation method and application of phellinus igniarius phenol A

Abstract

The invention relates to the technical field of fungus active ingredient extraction and medical application, and particularly provides a preparation method and application of phellinus igniarius phenol A. The preparation method comprises the following steps: extracting phellinus igniarius with an ethanol water solution and ethyl acetate in sequence to obtain an extract; the extract is subjected to silica gel column chromatography, Sephadex LH-20 (MeOH) column chromatography and ODS semi-preparative column separation in sequence, and the phellinus igniarius phenol A is obtained. In-vivo animal experiments prove that the phellinus igniarius phenol A has certain in-vitro inhibitory activity on six tumor cells, namely NCI-H460, MKN-45, 22Rv1, MCF7, SiHa and Hep 3B2.1-7, and particularly has the most remarkable in-vitro inhibitory effect on liver cancer Hep3B2.1-7 cells.

Description

Technical Field

[0001] The invention relates to the technical field of extraction of active ingredients of fungi and medical applications, and in particular to a preparation method and application of mulberry flavonoids. Background Art

[0002] Phellinus igniarius is a rare medicinal fungus with medicinal value such as anti-tumor, enhancing immunity, treating inflammation, anti-aging, lowering blood sugar, protecting the liver and repairing damage. Studies have shown that the effective medicinal ingredients in Phellinus igniarius are mainly polysaccharides, phenolic components, steroids and triterpenoid compounds. Tumors are a disease that seriously threatens human life and is one of the diseases that humans have not yet conquered. At present, the development and utilization of medicinal fungi with anti-tumor activity has become one of the directions for people to find new anti-tumor drugs, and it is also a research hotspot for scholars at home and abroad.

[0003] The Wnt / β-catenin signaling pathway plays an important role in the development of tumor growth and metastasis. Studies have shown that the Wnt / β-catenin signaling pathway is involved in the progression of liver cancer. In this pathway, β-catenin, as a core protein molecule, mediates the transmission of signals from the cytoplasm to the nucleus, and participates in the formation of a transcription complex in the nucleus to activate the transcription of downstream target genes. The complex formed by adenomatous polyposis coli (APC) will continuously degrade β-catenin in the cytoplasm and maintain its concentration at a low level. When APC is missing or deficient, β-catenin will accumulate in the cytoplasm and transfer to the nucleus, thereby activating downstream target genes including c-Myc and cyclin D1 to promote cell proliferation and induce the development of liver cancer. Inhibiting the Wnt / β-catenin signaling pathway of tumor cells provides ideas for finding new targets for tumor treatment.

[0004] The present invention explores a method for separating and purifying polyphenol compounds from Phellinus igniarius, and uses the obtained polyphenol compound, mulberry igniarius phenol A, to study its inhibitory activity on human breast cancer cells MCF7, human lung cancer cells NCI-H460, human gastric cancer cells MKN-45, human cervical cancer cells SiHa, human prostate cancer cells 22Rv1 and human liver cancer cells Hep 3B2.1-7, aiming to provide a certain reference and basis for screening anti-tumor drugs from medicinal fungi and studying the anti-tumor mechanism of medicinal fungi. Summary of the invention

[0005] The purpose of the present invention is to explore the separation method of active polyphenol components in Phellinus igniarius and its potential for preventing or treating cancer, thereby providing a preparation method and application of mulberry igniarius A. In vivo animal experiments have verified that mulberry igniarius A has certain in vitro inhibitory activity on six tumor cells, namely NCI-H460, MKN-45, 22Rv1, MCF7, SiHa and Hep 3B2.1-7, and the in vitro inhibitory effect on liver cancer Hep 3B2.1-7 cells is the most significant.

[0006] In order to achieve the above technical objectives, the technical solutions adopted in this application are as follows:

[0007] In a first aspect, the present invention provides a method for preparing mulberry flavonoids, comprising: extracting mulberry flavonoids with ethanol aqueous solution and ethyl acetate in sequence to obtain an extract; separating the extract by silica gel column chromatography, Sephadex LH-20 (MeOH) column chromatography and ODS semi-preparative column in sequence to obtain mulberry flavonoids; the structure of the mulberry flavonoids is shown in formula (I):

[0008]

[0009] Preferably, the volume concentration of the ethanol aqueous solution is 90%; and / or, the ethanol aqueous solution is used for extraction by mixing Phellinus igniarius with the ethanol aqueous solution and then subjecting the mixture to reflux extraction, more preferably 2 to 4 times, each time for 2.5 to 3.5 hours, and after extraction, concentrating to obtain the ethanol extract.

[0010] Preferably, the ethyl acetate extraction method is to mix the ethanol extract with an alkaline solution, separate the solid insoluble matter, neutralize it with an acidic solution, and then add ethyl acetate for extraction. More preferably, the extraction is performed 2 to 4 times. After the extraction is completed, the ethyl acetate extract is concentrated to obtain the extract.

[0011] Preferably, the silica gel column chromatography adopts the following gradient elution method: first, n-hexane and acetonitrile are used for gradient elution in a volume ratio of 100:0, 80:20, 50:50, 20:80, and 0:100, then methyl isobutyl ketone: acetone is used for gradient elution in a volume ratio of 50:50 and 0:100, and finally butanol: methanol is used for gradient elution in a volume ratio of 80:20, 50:50, 20:80, and 0:100; more preferably, the silica gel column chromatography process is combined with TLC and / or 254nm, 365nm ultraviolet fluorescence color development and / or ferric chloride color development to identify the isolate containing phenolic substances.

[0012] Preferably, the elution system of the ODS semi-preparative column separation is composed of a volume ratio of phase B to phase A of 30-40:60-70, wherein phase B is acetonitrile, and phase A is a mixture of water and acetic acid in a volume ratio of 1000:0.5-1.5; more preferably, the silica gel column chromatography process is combined with TLC and / or 254nm, 365nm ultraviolet fluorescence color development and / or ferric chloride color development to identify the isolate containing phenolic substances.

[0013] In a second aspect, the present invention provides the use of mulberry flavonoids A obtained by the preparation method described in the first aspect in the preparation of drugs for inhibiting Wnt / β-catenin signaling pathway.

[0014] In a third aspect, the present invention provides the use of mulberry phenol A obtained by the preparation method described in the first aspect in the preparation of anti-tumor drugs.

[0015] Preferably, the tumor includes tumors in the breast, lung, cervix, stomach, prostate, and liver.

[0016] In a fourth aspect, the present invention provides the use of mulberry phenol A obtained by the preparation method described in the first aspect in the preparation of anticancer drugs.

[0017] Preferably, the cancer includes breast cancer, lung cancer, cervical cancer, stomach cancer, prostate cancer, and liver cancer.

[0018] Compared with the prior art, the present invention has the following beneficial effects: the present invention extracts a high-purity polyphenol compound, mulberry flavonoids A, from the Chinese medicine Phellinus igniarius, and performs in vitro cell screening by the CCK-8 method to determine that mulberry flavonoids A has in vitro anti-tumor activity against six tumor cells, namely NCI-H460, MKN-45, 22Rv1, MCF7, SiHa and Hep 3B2.1-7, and has the most significant in vitro inhibitory effect on liver cancer Hep 3B2.1-7 cells. The present invention further verifies that mulberry flavonoids A has the effect of down-regulating Wnt / β-catenin pathway proteins through nude mouse in vivo transplant tumor experiments and western blot experiments, and preliminarily verifies the anti-cancer activity and mechanism of mulberry flavonoids A. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the HPLC preparation and separation diagram of mulberry flavonoids A in Example 1 of the present invention.

[0020] Figure 2 This is the identification diagram of Morinol A in Example 1 of the present invention, wherein a: fluorescence detection at 254nm; b: color development with ferric chloride; c: color development with vanillin sulfate.

[0021] Figure 3 This is the hydrogen nuclear magnetic resonance image of mulberry phenol A obtained in Example 1 of the present invention.

[0022] Figure 4 This is the carbon nuclear magnetic resonance image of mulberry flavonoids A obtained in Example 1 of the present invention.

[0023] Figure 5 This is a graph showing the inhibition of tumor cell proliferation in vitro by mulberry flavonoids A in Example 2 of the present invention.

[0024] Figure 6 Figure 3 shows the effect of mulberry flavonoids A on the Hep 3B2.1-7 transplanted tumor model in Example 3 of the present invention, wherein Figure A is a curve showing the change of body weight in different experimental groups over the number of days; Figure B is a curve showing the change of tumor volume in different experimental groups over the number of days; and Figure C is the size of tumors in different experimental groups.

[0025] Figure 7 Figure 4 is a graph showing the effect of mulberry flavonoids A on the expression of Wnt / β-catenin pathway proteins in Example 4 of the present invention, wherein Figure A is an immunoblot of Wnt / β-catenin pathway proteins, Figure B is a histogram of the relative expression of APC protein versus concentration, Figure C is a histogram of the relative expression of β-catenin protein versus concentration, Figure D is a histogram of the relative expression level of c-Myc protein versus concentration, and Figure E is a histogram of the relative expression of cyclin D1 protein versus concentration. DETAILED DESCRIPTION

[0026] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it.

[0028] Example 1: Isolation, preparation and structural confirmation of the compound Morinol A

[0029] (1) Separation and preparation

[0030] Phellinus linteus slices (1kg) are extracted 3 times with 10L of 90% ethanol through a multifunctional vacuum extraction and concentration tank at 60°C, each time for 3 hours, and the combined extracts are decompressed and distilled until no extract residues are left, to obtain about 103g of Phellinus linteus ethanol extract. The ethanol extract is fully dissolved with dilute alkaline water, filtered, and the filtrate is neutralized with an equivalent amount of dilute acid water, and then extracted three times with ethyl acetate, and the ethyl acetate extract is combined, concentrated to remove ethyl acetate, and 52.6 grams of ethyl acetate extract is obtained. In this separation step, the present invention has previously found in the exploration of extraction solvents that it is difficult to separate the polyphenol compounds therein by using only ethanol, methanol, propanol, ethyl acetate, acetone, and tetrahydrofuran with greater polarity for extraction, so a multi-stage extraction method is adopted, the first extraction uses ethanol with greater polarity (methanol is more toxic and is not considered in the end), and the second extraction uses ethyl acetate with relatively smaller polarity. This is because the ethanol extract contains many fatty acids and sugar compounds, while the phenolic compounds we need are mostly medium polar compounds, which can be obtained by extraction with ethyl acetate. We also tried acetone, chloroform, and dichloromethane, several medium polar solvents, but ultimately failed to obtain phenolic compounds. Therefore, ethyl acetate was used to extract the ethanol extract to obtain the final extract.

[0031] The ethyl acetate extract was mixed with an appropriate amount of 100-200 mesh silica gel and then loaded onto a silica gel column. First, n-hexane:acetonitrile (100:0, 80:20, 50:50, 20:80, 0:100) was used for gradient elution, the fraction was enriched once every 2 L, and 8 L was taken for each solvent system; then, methyl isobutyl ketone:acetone (50:50, 0:100) was used for gradient elution, the fraction was enriched once every 2 L, and 8 L was taken for each solvent system; finally, butanol:methanol (80:20, 50:50, 20:80, 0:100) was used for gradient elution, the fraction was enriched once every 2 L, and 8 L was taken for each solvent system; according to TLC and 254nm and 365nm ultraviolet fluorescence color development, combined with phenolic compound detection reagent ferric chloride color development, similar components were combined to obtain three separation parts Fr.1-Fr.3.

[0032] In the initial stage of experimental exploration, the present invention adopted: (1) n-hexane and acetonitrile were gradient eluted in the volume ratio of 100:0, 80:20, 50:50, 20:80, and 0:100; (2) methanol and acetonitrile were gradient eluted in the volume ratio of 100:0, 80:20, 50:50, 20:80, and 0:100; (3) methanol and acetonitrile were gradient eluted in the volume ratio of 100:0, 80:20, 50:50, 20:80, and 0:100; none of the target separation sites were obtained. The gradient elution method was further adjusted as follows: (4) n-hexane and acetonitrile were gradient eluted in the following volume ratios: 100:0, 80:20, 50:50, 20:80, 0:100, and then butanol and methanol were gradient eluted in the following volume ratios: 80:20, 50:50, 20:80, 0:100; (5) methanol and acetonitrile were gradient eluted in the following volume ratios: 100:0, 80:20, 50:50, 20:80, 0:100, and then methyl isobutyl ketone and acetone were gradient eluted in the following volume ratios: (6) Methanol and acetonitrile were gradient eluted in the volume ratio of 100:0, 80:20, 50:50, 20:80, 0:100, and then methyl isobutyl ketone and acetone were gradient eluted in the volume ratio of 50:50, 0:100. None of them obtained the target separation site. Methods (4) to (6) all obtained one separation site, but no polyphenol compounds were separated in the end. The gradient elution method was further adjusted as follows: (7) n-hexane and acetonitrile were gradient eluted in the volume ratios of 100:0, 80:20, 50:50, 20:80, and 0:100, and then methyl isobutyl ketone and acetone were gradient eluted in the volume ratios of 50:50 and 0:100, respectively, to obtain two separation parts, but no polyphenol compounds were finally separated.

[0033] Fr.2 (9.6 g) was separated by Sephadex LH-20 (MeOH) column chromatography to obtain 4 fractions (Fr.2.1-Fr.2.4). Fr.2.4 (280 mg) was separated by HPLC (elution system: B:A=35:65, A phase: (water: acetic acid 1000:1), B phase: acetonitrile, retention time: 17 min) using an ODS semi-preparative column, and the monomer compound mulberry yellow phenol A (164 mg) was obtained by combining ferric chloride colorimetric detection. In the early stage of experimental exploration, the present invention sequentially used different analytical conditions to perform HPLC analysis on Fr.2.4. First, we used phase B (acetonitrile): phase A (water: acetic acid 1000:1) in a volume ratio of 5:95-100:0 for gradient analysis for 60 min, and then used phase B for analysis for 30 minutes. We found that the retention time of the main components in the HPLC analysis chart was within the range of B:A concentration gradient of 20:80-40:60, so we used B:A = 20:80, 25:75, 30:70, 35:65, and 40:60 volume concentrations for isocratic analysis. Finally, we found that when the B:A concentration was 35:65, all components in Fr.2.4 had good separation and sharp peaks. The HPLC liquid phase diagram is shown in the figure. Figure 1 As shown, TLC identification Figure 2 As shown, we finally determined the elution system to be B:A=35:65.

[0034] (2) Structural confirmation

[0035] The obtained mulberry yellow phenol A was identified by hydrogen nuclear magnetic resonance and carbon nuclear magnetic resonance. Figure 3 and Figure 4 As shown, the structural information provided in the spectrum is: amorphous powder, C 19 H 12 O7, 1 H-NMR (600MHz, DMSO-d6) δ: 7.39 (m, H-7, 2'), 7.20 (d, J = 9Hz, H-9), 7.07 (s, H-3, 5'), 6.97 (s, H-4), 6.77-6.81 (m, H-6, 12); 13C-NMR(151MHz,DMSO-d6)δ:162.80(C-3),158.05(C-5),157.88(C-1),149.0 6(C-6'),147.52(C-11),146.16(C-10),145.74(C-3'),144.57(C-4'),134.0 4(C-7),126.95(C-8),120.40(C-13),116.48(C-6),115.92(C-12),113.90(C -1'),113.56(C-9),104.64(C-2'),103.83(C-2),98.84(C-5'),95.90(C-4). The above spectrum data is consistent with the data reported in the literature [Phenolic compounds with NF-jBinhibitory effects from the fungus Phellinus baumii.doi:10.1016 / j.bmcl.2011.04.025], so the structure is determined to be phellibauminA, which is a phenolic compound isolated from Phellinus baumii and named as Phellinus flavonoids A. The structure is as follows:

[0036] Structural formula of mulberry flavonoids.

[0037] Example 2: Screening of in vitro anti-tumor activity of mulberry flavonoids

[0038] In this example, several malignant tumor cells were selected to investigate the in vitro anti-tumor activity of the polyphenol compound, as follows:

[0039] 1. Culture of tumor cells:

[0040] Human breast cancer cells MCF7, human lung cancer cells NCI-H460, and human cervical cancer cells SiHa were purchased from the Chinese Academy of Sciences Cell Bank, and human gastric cancer cells MKN-45, human prostate cancer cells 22Rv1, and human liver cancer cells Hep 3B2.1-7 were purchased from the Wuhan University Collection Center. Sterile DMEM and RPMI 1640 cell culture media containing 10% FBS and 1% double antibody solution were used, of which NCI-H460, MKN-45, and 22Rv1 cells were cultured in RPMI 1640 medium, and MCF7, SiHa, and Hep3B2.1-7 cells were cultured in DMEM culture medium in a 5% CO2, 37°C cell culture incubator environment.

[0041] 2. Effect of Morinol A on the Inhibitory Activity of Tumor Cells in Vitro:

[0042] Tumor cells in the logarithmic growth phase were digested and counted, and the tumor cells were inoculated in a 96-well plate, with the number of cells inoculated per well set to 7 × 10 3 An appropriate amount of PBS was added to the edge wells to prevent edge effects. The 96-well plate was then placed in a cell culture incubator for culture. After culturing for 24 hours until the cells adhered to the wall, the original culture medium was discarded and the drug-containing culture medium was added. The concentrations of the active monomer of Phellinus igniarius, mulberry flavonoids, were set to 0, 6.25, 12.5, 25, 50 and 100 μM, and the negative control group was added with a drug-free culture medium. The 96-well plate was then placed in a cell culture incubator for continued culture. After 48 hours, the drug-containing culture medium was discarded, and 110 μL of culture medium containing 10 μL CCK-8 reagent was added to each well; at the same time, PBS was discarded from the edge wells, and 110 μL of culture medium containing 10 μL CCK-8 reagent was also added to each well as a blank control group. It was then incubated at 37°C. After incubation for 2 hours, the 96-well plate was placed in an enzyme reader to measure the absorbance at a wavelength of 450 nm. The cell proliferation inhibition rate was calculated using the following formula:

[0043] Cell proliferation inhibition rate (%) = [1-(A sample-A blank) / (A control-A blank)] × 100%

[0044] A sample is the absorbance of the drug group, A blank is the absorbance of the blank control group, and A control is the absorbance of the negative control group.

[0045] Table 1 IC50 (μM) values ​​of mulberry phenol A for the inhibition of proliferation of various tumor cells

[0046] MCF7 NCI-H460 MKN-45 SiH Hep 3B2.1-7 22Rv1 Phellibaumin A 37.51±4.36 45.62±2.19 59.89±10.16 34.60±5.05 22.34±6.43 39.72±6.50

[0047] Through in vitro cell screening with CCK-8 experiments, we can intuitively obtain the inhibitory activity of mulberry flavonoids on various tumor cells in vitro. It was found that mulberry flavonoids had certain in vitro inhibitory activity on six tumor cells, among which the in vitro inhibitory effect on liver cancer Hep3B2.1-7 cells was the most significant, with an IC50 value of 22.34±6.43μM. Mulberry flavonoids have the effect of inhibiting tumor cell proliferation in vitro ( Figure 5 ).

[0048] Example 3: Antitumor activity of mulberry flavonoids in nude mice with Hep 3B2.1-7 transplanted tumor model

[0049] In view of the experimental results of Example 2, the in vivo effect of mulberry flavonoids A on liver cancer was further investigated in this example, as follows:

[0050] 1. Cultivation of nude mice:

[0051] SPF grade BALB / c-nude mice, 3-4 weeks old, weighing 10-12g, male, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and raised in the Experimental Animal Center of Anhui Medical University according to SPF animal grade conditions. Feeding conditions: ambient temperature at 20-26℃, humidity at 40%-70%, free access to water and feed, and the water and feed consumed, as well as the cages and bedding in contact with them, were sterilized by high temperature and high pressure. All nude mice were fed adaptively for one week before the model was established.

[0052] 2. Establishment of transplanted tumor model:

[0053] Hep 3B2.1-7 cells were collected and counted, and pre-cooled PBS was added after centrifugation to prepare a single cell suspension, and the concentration was adjusted to 1×107 cells / mL. The single cell suspension was aliquoted and sealed, and transferred to the SPF-level experimental animal center in an ice box. In the clean bench, the cell suspension was drawn up with an insulin syringe, and the inoculation dose was 200 μL / animal (approximately 2.0×10 6 The mice were inoculated subcutaneously in the axilla of the right forelimb with a slow push injection of 100 cells. After inoculation, the mice were fed under normal conditions, and the growth of the transplanted tumors was observed and recorded daily.

[0054] 3. Effects of Morinol A on Tumor-transplanted Nude Mice:

[0055] When the volume of the subcutaneous transplanted tumor in nude mice grows to 100 mm 3 The transplanted tumor model mice were randomly divided into the following groups: model control group (normal saline), positive control group (CDDP 10 mg / kg), low-dose group (25 mg / kg), medium-dose group (50 mg / kg) and high-dose group (100 mg / kg). All drug solutions were prepared with normal saline on the day of administration. The positive control group was intraperitoneally injected once every 2 days, and the other groups were gavage-administered once a day. The body weight and transplanted tumor volume of nude mice were measured every two days during the administration period, and the nude mouse body weight time growth curve and tumor volume time growth curve were drawn ( Figure 6 The drug was administered continuously until the tumor volume of the model control group reached about 1000 mm 3 About (the tumor size of each experimental group is as follows Figure 6 C), nude mice were killed by cervical dislocation to obtain tumor tissues, and the tumor weight was weighed and recorded. The tumor inhibition rate was calculated according to the following formula:

[0056] Tumor inhibition rate (%) = (tumor weight of model group - tumor weight of drug administration group) / tumor weight of model group control group × 100%

[0057] Table 2 Tumor inhibition rate of mulberry phenol A on Hep 3B2.1-7 transplanted tumor model in nude mice

[0058]

[0059] According to the changes in tumor volume and weight of each group before and after administration, the in vivo anti-tumor activity of the drug can be intuitively determined. The experimental results show that mulberry flavonoids can significantly inhibit the growth of tumor volume and weight of nude mice with liver cancer Hep 3B2.1-7 transplanted tumors, exert anti-tumor effects in vivo, and the inhibitory effect is obviously dose-dependent; at the same time, the weight of nude mice is stable before and after administration, indicating that mulberry flavonoids have no obvious toxic side effects ( Figure 6 ).

[0060] Example 4: Effect of Morinol A on the Expression of Wnt / β-catenin Pathway Proteins

[0061] This example is to explore the therapeutic mechanism of mulberry phenol A against liver cancer, as follows:

[0062] 1. Preparation of protein samples:

[0063] Hep 3B2.1-7 cells were collected and counted, and seeded in a six-well plate, with the number of cells seeded per well set to 1×10 5 After 24 hours of culture, add culture medium containing mulberry yellow phenol A (concentrations set to: 0, 5, 10, 20 μM) and continue to culture for 48 hours. At the end of the culture, discard the culture medium and wash with cold PBS. Add 0RIPA containing PMSF and phosphatase inhibitors on ice to fully lyse for 15 minutes. Scrape the cells with a cell scraper, collect and transfer them to a pre-cooled centrifuge tube, and centrifuge at 4°C, 12000rpm for 15 minutes. Collect the supernatant, add SDS-PAGE protein loading buffer, and boil in a constant temperature water bath at 100°C for 10 minutes to obtain the desired protein sample.

[0064] 2. Detection of protein expression:

[0065] Use a 10% PAGE gel rapid preparation kit to prepare the gel, add the prestained protein marker and sample protein to the gel wells, and load 10 μg of protein per well. Set the electrophoresis condition to a constant voltage of 80V for 30 minutes. After the marker begins to separate, adjust the electrophoresis condition to a constant voltage of 120V for 60 minutes and end the electrophoresis. Then, under ice bath conditions, set a constant current of 220mA and transfer the protein bands to the PVDF membrane for 120 minutes. After the transfer, use skim milk to block at room temperature for 2 hours, incubate with the primary antibody at 4°C overnight, and then incubate with the secondary antibody at room temperature for 1 hour. Use the newly configured developer to evenly apply it on the PVDF membrane, expose it to the developer for imaging observation, and the results are as follows: Figure 7 As shown in AE.

[0066] After 48h of treatment with mulberry flavonoids, the expression levels of β-catenin, c-Myc, cyclin D1 and Wnt3a proteins in Hep 3B2.1-7 cells were downregulated as the concentration of mulberry flavonoids increased; while the expression level of APC protein was upregulated as the concentration of mulberry flavonoids increased. This indicates that the Wnt / β-catenin signaling pathway in Hep 3B2.1-7 cells is inhibited by mulberry flavonoids ( Figure 7 (Medium AE).

[0067] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing mulberry phenol A, characterized in that: include: Phellinus linteus was extracted with ethanol aqueous solution and ethyl acetate in sequence to obtain an extract; the extract was separated by silica gel column chromatography, Sephadex LH-20 (MeOH) column chromatography and ODS semi-preparative column in sequence to obtain mulberry linteus A; the structure of the mulberry linteus A is shown in formula (I):

2. The preparation method according to claim 1, characterized in that: The volume concentration of the ethanol aqueous solution is 90%; and / or, the ethanol aqueous solution is used for extraction by mixing Phellinus igniarius with the ethanol aqueous solution and then performing reflux extraction, preferably 2 to 4 times, each extraction taking 2.5 to 3.5 hours, and concentrating after the extraction to obtain an ethanol extract.

3. The method according to claim 2, characterized in that The method of using the ethyl acetate extraction is to mix the ethanol extract with an alkaline solution, separate the solid insoluble matter, neutralize it with an acidic solution, and then add ethyl acetate for extraction, preferably extracting 2 to 4 times. After the extraction is completed, concentrate to obtain the ethyl acetate extract.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The silica gel column chromatography adopts the following gradient elution method: firstly, n-hexane and acetonitrile are used for gradient elution in a volume ratio of 100:0, 80:20, 50:50, 20:80 and 0:100, then methyl isobutyl ketone:acetone are used for gradient elution in a volume ratio of 50:50 and 0:100, and finally butanol:methanol are used for gradient elution in a volume ratio of 80:20, 50:50, 20:80 and 0:

100. Preferably, the silica gel column chromatography process is combined with TLC and / or 254nm, 365nm ultraviolet fluorescence color development and / or ferric chloride color development to identify the isolate containing phenolic substances.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The elution system of the ODS semi-preparative column separation is composed of a volume ratio of phase B to phase A of 30-40:60-70, wherein phase B is acetonitrile, and phase A is a mixture of water and acetic acid in a volume ratio of 1000:0.5-1.5; preferably, the silica gel column chromatography process is combined with TLC and / or 254nm, 365nm ultraviolet fluorescence color development and / or ferric chloride color development to identify the isolate containing phenolic substances.

6. Use of mulberry flavonoids A obtained by the preparation method according to any one of claims 1 to 5 in the preparation of drugs for inhibiting Wnt / β-catenin signaling pathway.

7. Use of mulberry flavonoids A obtained by the preparation method according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: The tumors include those in the breast, lung, cervix, stomach, prostate, and liver.

9. Use of mulberry flavonoids A obtained by the preparation method according to any one of claims 1 to 5 in the preparation of anticancer drugs.

10. The use according to claim 9, characterized in that: The cancers include breast cancer, lung cancer, cervical cancer, stomach cancer, prostate cancer, and liver cancer.

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