Application of granatanine in preparation of antitumor drugs

Abstract

The invention provides an application of punicalin (PNC) in preparation of antitumor drugs, and belongs to the technical field of biological medicines related to tumor diseases. The tumor proteins TNC and FLNC can jointly promote endothelial-mesenchymal transition (EndMT). The PNC is a bimolecular targeting natural inhibitor of tumor proteins TNC and FLNC. The PNC can inhibit the expression of EndMT related protein, multidrug resistance related membrane protein and collagen related protein through targeted inhibition of TNC and FLNC, so that the sensitivity of drug-resistant tissues to chemotherapeutic drugs is recovered. The PNC can be developed into a universal EndMT and collagen pathway targeting drug for reversing tumor drug resistance, combined chemotherapy drugs are developed from the aspects of tumor microenvironment endothelial cells, ECM deposition and the like so as to improve drug sensitivity, and the PNC has important clinical significance in tumor resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine related to tumor diseases, and in particular relates to the application of pomegranate peel tannin in the preparation of anti-tumor drugs. Background Art

[0002] Therapeutic resistance has always been the main reason affecting the prognosis of cancer patients and driving tumor recurrence. In recent years, studies have found that the interaction between the tumor microenvironment (TME) and tumor cells plays an important role in the formation of drug resistance. TME is a complex ecosystem composed of a variety of immune cells, tumor-associated endothelial cells (TECs), cancer-associated fibroblasts (CAFs) and extracellular matrix (ECM), which plays an important role in tumor immune escape, invasion, treatment resistance and other processes.

[0003] TECs are considered to be a key stromal cell type in the TME, playing a core role in tumor angiogenesis and blood supply. The phenotypic plasticity of TECs has gradually attracted attention, and the endothelial-mesenchymal transition (EndMT)-related process is a key event in tumor progression. During EndMT, vascular endothelial cells lose endothelial characteristics and acquire a mesenchymal phenotype under external stimuli, which is considered to be one of the main sources of CAFs. EndMT-derived fibroblasts and their derived ECM play an important role in cancer therapy resistance, including the formation of a dense ECM physical barrier around and throughout the tumor, hindering drug diffusion and transport; ECM proteins adhere to cancer cells to evade chemotherapy; crosstalk with immune cells to form a tumor immunosuppressive microenvironment, stimulate paracrine factors, and promote cancer cell growth. Therefore, EndMT has become an emerging potential therapeutic target for tumor treatment.

[0004] Punicalin (PNC) is a hydrolyzable tannin isolated from pomegranate peel, pomegranate leaves, pomegranate juice and other parts, which has strong pharmacological activities such as antioxidant, anti-liver damage, antibacterial, antiviral and anti-inflammatory. However, there is no relevant report on whether PNC has anti-tumor effect and directly acts on target protein. Summary of the invention

[0005] The purpose of the present invention is to provide application of pomegranate peel tannin in preparing anti-tumor drugs.

[0006] The invention provides application of pomegranate peel tannin in preparing anti-tumor drugs.

[0007] Preferably, the anti-tumor drug includes an anti-tumor drug that improves chemotherapy sensitivity.

[0008] The invention provides the use of pomegranate peel tannin combined with chemotherapy drugs in the preparation of anti-tumor drugs.

[0009] Preferably, the chemotherapy drug comprises temozolomide or etoposide.

[0010] Preferably, the tumor comprises a glioma or a glioblastoma.

[0011] The present invention provides the use of pomegranate peel tannin in preparing an inhibitor of tumor protein TNC and / or tumor protein FLNC; the amino acid sequence of the TNC is shown as SEQ ID NO.1; the amino acid sequence of the FLNC is shown as SEQ ID NO.2.

[0012] The invention provides application of pomegranate peel tannin in preparing an endothelial-mesenchymal transition inhibitor.

[0013] The invention provides the use of pomegranate peel tannin in preparing a protein expression inhibitor; the protein comprises one or more of endothelial-mesenchymal transition-related proteins, multidrug resistance-related membrane proteins and collagen-related proteins.

[0014] The invention provides an anti-tumor drug, comprising pomegranate peel tannin and a chemotherapy drug.

[0015] Preferably, the chemotherapy drug comprises temozolomide or etoposide.

[0016] The present invention provides the use of pomegranate peel tannin in the preparation of anti-tumor drugs. Tumor proteins TNC and FLNC can jointly promote EndMT. PNC is a bimolecular targeted natural inhibitor of tumor proteins TNC and FLNC. PNC can inhibit the expression of EndMT-related proteins, multidrug resistance-related membrane proteins and collagen-related proteins by targeted inhibition of TNC and FLNC, thereby restoring the sensitivity of resistant tissues to chemotherapy drugs. PNC can be developed into a universal drug that targets EndMT and collagen pathways to reverse tumor resistance, and combined chemotherapy drugs can be developed from the perspectives of tumor microenvironment endothelial cells, ECM deposition, etc. to improve drug sensitivity, which has important clinical significance for anti-tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1Indicates that CAFs infiltration increases in recurrent and resistant GBM tissues; a-b are IHC staining of CAFs markers α-SMA and COL1A1 in primary and recurrent GBM sections; c is a statistical result of the number of α-SMA and COL1A1 positive cells; d is the IHC staining result of α-SMA in normal brain tissue, TMZ-sensitive tissue and TMZ-resistant tissue;

[0019] Figure 2 Collagen pathway enrichment after HCMECs co-cultured with drug-resistant cells; a is a schematic diagram of protein profile and RNA-seq sequencing of supernatant and total cell RNA collected after co-culture of human brain microvascular endothelial cells (HCMECs) with TMZ-sensitive (S) and TMZ-resistant (R) cells; b is a Venn diagram of differentially expressed proteins (DEPs) and differentially expressed genes (DEGs); c is GO enrichment analysis of differentially expressed proteins;

[0020] Figure 3 To further demonstrate the correlation between TNC and FLNC and CAFs infiltration and the expression of ECM proteins COL1A1 and FN1;

[0021] Figure 4 Indicates that TNC / FLNC promotes EndMT and ECM deposition; a is TMZ-R cell culture supernatant promoting HCMECsEndMT phenotype; b is TNC and FLNC recombinant proteins promoting EndMT; c is the half-inhibitory concentration of ETO and TMZ in HCMEC cells; d is Western blot detection of TNC, FLNC, and TGF-β expressions in HCMEC co-cultured with TMZ-S / R and under the action of ETO; e is HCMEC co-cultured with TMZ-S / R and under the action of FLNC / TNC (TF) recombinant proteins. Expression; f is ELISA detection of COL1A1, FN1, TNC, and FLNC secretion in HCMECs under TMZ-S / R and ETO stimulation;

[0022] Figure 5Figure 1 is the result of natural small molecule screening and functional verification targeting TNC / FLNC; a is the prediction of active sites of FLNC protein and TNC protein respectively using Sitemap software, and the comparison results after aligning the two proteins; b is the TOP3 target compounds of FLNC and TNC; c is the interaction mode of MOL009272 (Punicalin, PNC) with FLNC and TNC; the 3D diagram shows that the protein and the different hydrogen bond donors / acceptors of the compound form a certain number of hydrogen bond interactions (red), π-cation interactions (green) and salt bridge interactions (yellow), and the specific interacting residues are shown in the figure. In the figure, the dotted line next to the force is the bond length in angstroms; in the 2D figure, hydrogen bonds are represented by purple arrows, and cation-π stacking is represented by red lines; the amino acid colors are red for acidic amino acids, green for hydrophobic amino acids, cyan for polar amino acids, and blue for basic amino acids; d is the half-inhibitory concentration of the TOP3 target compounds Chelidimerine (CHE), PNC, and Neoprzewaquinone A (NA) in HCMEC cells; e is the expression verification of CHE, PNC, and NA on TNC / FLNC and collagen-related proteins, EndMTMarker proteins, and drug resistance-related membrane proteins;

[0023] Figure 6 It indicates that animal experiments have verified that PNC can reverse drug resistance; using the TMZ-R cell tumor-bearing mouse model, the tumor growth curves of each group of mice after treatment are shown in Figure a; Figure b is the tumor size; and Figure c shows the tumor weight. DETAILED DESCRIPTION

[0024] The invention provides application of pomegranate peel tannin (PNC) in preparing anti-tumor drugs.

[0025] In the present invention, the chemical structure of the PNC is shown in Formula 1, which is as follows:

[0026]

[0027] In the specific implementation of the present invention, the pomegranate peel tannin was purchased from Wuhan Tianzhi Biotechnology Co., Ltd.

[0028] In the specific implementation of the present invention, the anti-tumor drug includes an anti-tumor drug that improves chemotherapy sensitivity.

[0029] The present invention also provides the use of pomegranate peel tannin combined with chemotherapy drugs in the preparation of anti-tumor drugs.

[0030] In a specific implementation of the present invention, the chemotherapy drug includes temozolomide or etoposide.

[0031] In the present invention, the pomegranate peel tannin can reverse tumor resistance.

[0032] In the specific implementation of the present invention, the tumor includes glioma or glioblastoma; the tumor includes a drug-resistant tumor; and the drug-resistant tumor is tolerant to chemotherapy drugs.

[0033] In the specific implementation of the present invention, the dosage form of the anti-tumor drug is an oral preparation.

[0034]

[0035] TNC and FLNC have certain homology. The present invention combines dry and wet experiments to find that TNC and FLNC are positively correlated with tumor CAFs infiltration and ECM deposition, and can promote endothelial-mesenchymal transition (EndMT) and collagen pathway activation, thereby inhibiting chemotherapy sensitivity. PNC is a bimolecular targeted natural inhibitor of tumor proteins TNC and FLNC. PNC inhibits the expression of EndMT-related proteins, multidrug resistance-related membrane proteins, and collagen-related proteins by targeted inhibition of TNC and FLNC, thereby inhibiting EndMT and ECM deposition, restoring the sensitivity of resistant tissues to chemotherapeutic drugs, and can be applied to the targeted treatment of tumors to play a role in reversing drug resistance.

[0036] PNC can be developed into a universal drug that targets EndMT and collagen pathways to reverse tumor resistance, and combined chemotherapy drugs can be developed from the perspectives of tumor microenvironment endothelial cells, ECM deposition, etc. to improve drug sensitivity, which has important clinical significance for anti-tumor.

[0037] The present invention also provides the use of pomegranate peel tannin in the preparation of endothelial-mesenchymal transition (EndMT) inhibitors.

[0038] The present invention also provides the use of pomegranate peel tannin in the preparation of protein expression inhibitors; the protein includes one or more of endothelial-mesenchymal transition-related proteins, multidrug resistance-related membrane proteins and collagen-related proteins.

[0039] In the present invention, PNC targets EndMT and collagen pathway-related proteins.

[0040] The invention also provides an anti-tumor drug, comprising pomegranate peel tannin and a chemotherapy drug.

[0041] In the present invention, the combination of PNC and chemotherapeutic drugs can be used to reverse the drug resistance of tumors to chemotherapeutic drugs.

[0042] In a specific implementation of the present invention, the chemotherapy drug includes temozolomide or etoposide.

[0043] In the specific implementation of the present invention, based on mice, the dosage of the pomegranate peel tannin is 40 mg / kg; the dosage of the temozolomide (TMZ) is 20 mg / kg.

[0044] In order to further illustrate the present invention, the application of the pomegranate peel tannin provided by the present invention in the preparation of anti-tumor drugs is described in detail below in combination with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] The collected glioma tissue chip samples including primary and recurrent samples and mouse resistant tissue sections were subjected to immunohistochemical staining with α-SMA antibody (Proteintech, 14395-1-AP) and COL1A1 (Proteintech, 14695-1-AP):

[0047] The slice samples were sequentially immersed in xylene I for 5 min, replaced with xylene II for 5 min, anhydrous ethanol for 3 min, 95% ethanol for 3 min, 80% ethanol for 3 min, 70% ethanol for 3 min, and tap water for 3 min for dewaxing and hydration, then placed in antigen repair solution (citrate buffer, pH 6.0) at 95-98 ° C for 15 min, cooled naturally to room temperature for antigen repair, and washed 3 times with PBST buffer, each time for 3 min. Goat serum blocking solution was added dropwise and kept at room temperature for 30 min. The blocking solution was shaken off, and the primary antibody (α-SMA / COL1A1, 1:100) was added and kept at 4 ° C overnight. Wash 3 times with PBST buffer, 3 minutes each time, add corresponding secondary antibody, room temperature for 1-2 hours, wash 3 times with PBST buffer, 3 minutes each time, DAB color development (DAB horseradish peroxidase color development kit, Bio-Tech), rinse with tap water for 10 minutes, counterstain with hematoxylin (Soler Bio) for about 30 seconds, quickly rinse with tap water, dehydrate with alcohol gradient (70%, 80%, 95%, 100%, xylene I, xylene II), and seal with neutral resin. Take pictures with an inverted microscope. Staining results are shown in Figure 1 , showing that α-SMA and COL1A1 were highly expressed in patients with recurrent glioma and in drug-resistant tissues.

[0048] Example 2

[0049] 1. Cell lines

[0050] Human brain microvascular endothelial cells (HCMECs) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai). U87 was used to construct an in vivo acquired TMZ (temozolomide) resistance model (TMZ-R) and a parallel control sensitive group (TMZ-S). The obtained TMZ-R and TMZ-S cells were used for subsequent experiments.

[0051] 2. Cell Recovery

[0052] Take out the frozen cells from the -80℃ refrigerator and thaw them in a 37℃ water bath. After the cell freezing solution is completely dissolved, centrifuge at 1000rpm for 5min and discard the supernatant. Add 1ml of complete culture medium to resuspend the cell pellet, mix it by pipetting and inoculate it in a culture dish, shake it well, and place it at 37℃ and 5% CO. 2 Culture in incubator.

[0053] 3. Cell Passaging

[0054] Take cells with suitable density and good growth status, remove the culture medium and wash three times with PBS, then discard the PBS, add appropriate amount of trypsin to the culture dish, digest at room temperature for a suitable time, and observe that the cells are dispersed into single, sand-like flow under the microscope, and add an equal volume of complete culture medium to terminate the digestion. Transfer the fully digested cell suspension to a new EP tube, centrifuge at 1000rpm for 5min, and discard the supernatant. Resuspend the cell pellet with 1ml of complete culture medium, then pipet and mix well and add to a new culture dish, add appropriate amount of complete culture medium, and place at 37℃ and 5% CO 2 Cultivated in environment.

[0055] 4. Cell Counting

[0056] Rinse the counting plate and cover glass with double distilled water, wipe the counting plate with 75% ethanol solution, and dry it for later use. Slowly add 10μl of cell suspension from the edge of the counting plate, and use the siphon principle to fill the counting pool. After the cells spread, settle, and fill the pool, observe and count under a microscope.

[0057] 5. Cell Cryopreservation

[0058] Take the cells that need to be frozen in the logarithmic phase and place them in a test tube. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and collect the cell precipitate. Add an appropriate amount of cell freezing solution to the test tube, mix it by pipetting, and then divide it into cryopreservation tubes and store it in a -80℃ refrigerator.

[0059] 6. Sequencing Analysis

[0060] After HCMEC was co-cultured with TMZ-S and TMZ-R cells for 48 hours, serum-free medium was replaced and cultured for another 48 hours. Cell supernatants and total cell RNA were collected. Label free proteome sequencing and RNA-seq sequencing were performed on the samples by Hangzhou Lianchuan Biology to obtain differentially expressed proteins (DEPs) and differentially expressed genes (DEGs). Then, the upregulated DEGs were plotted into a heat map using the "ggplot2" software package (https: / / www.xiantaozi.com / ). In order to study the functions of upregulated DEGs, Gene Ontology (GO) enrichment analysis was performed online. The analysis results are shown in Figure 2 , it can be seen that the collagen pathway and cytokine-mediated pathways are enriched after HCMECs cells are co-cultured with drug-resistant cells.

[0061] 7. Immunofluorescence (IF) staining

[0062] The cells were inoculated and washed with PBS three times, 5 minutes each time. 4% paraformaldehyde was used to fix the cells at room temperature for 15 minutes, and then washed with PBS. 0.3% TritonX-100 was used to permeabilize the cells at room temperature for 20 minutes, and then washed with PBS. 5% bovine serum albumin (BSA) was used to block the cells at room temperature for 1 hour. Then the primary antibody was added and incubated at 4°C overnight. The primary antibody was discarded the next day, and then the cells were washed with PBS. The fluorescent secondary antibody was added and incubated at room temperature in the dark for 1 hour, and then washed with PBS. The cell nuclei were counterstained with Hoechst, and the cells were sealed with sealing agents and photographed under a microscope.

[0063] 8. In vitro efficacy study of etoposide (ETO) and TMZ

[0064] ETO (Etoposide) and TMZ (Temozolomide) are both commonly used anti-tumor chemotherapy agents. ETO mainly inhibits DNA replication by inhibiting topoisomerase II. TMZ is a DNA alkylating agent, and its active product MTIC (3-methyl-(triazine-1-)imidazole-4-carboxamide) alkylates the oxygen atom at the 6th position of guanine and the nitrogen atom at the 7th position on the DNA molecule, and exerts a cytotoxic effect through mismatch repair of the methylated adduct.

[0065] Take 200 μL (5000 cells) of HCMECs cell suspension in the logarithmic growth phase and place them in 96-well culture plates, which are then placed in an incubator (37°C, 5% CO 2 ) After 48 hours of culture, the culture medium was discarded, and different concentrations of ETO and TMZ were first added for 48 hours. Another group of cells without drug treatment was set up as a control, and 6 replicates were set up in parallel. After that, the drug-containing culture medium was removed, and 200 μL of blank culture medium containing 10 μL CCK-8 solution was added to the 96-well plate, and the incubation continued for 2 hours. The absorbance was measured with an enzyme marker at a wavelength of 450nm, and the cell survival rate and IC50 value were calculated. The concentration with an ETO cell inhibition rate of 10% to 20% was selected as the safe concentration for subsequent cell experiments.

[0066] 9. Western Blot

[0067] Wash the glass plate and then dry it in an oven. Prepare a separation gel of appropriate concentration and add it to the glass plate. Immediately and slowly add the concentrated gel and insert the comb. After the concentrated gel is solidified, remove the comb. Prepare the electrophoresis solution and the rapid transfer solution and keep them in a refrigerator at four degrees for later use. Load 20 to 40 μg of protein per well. First, perform constant voltage electrophoresis at less than 100V. After the indicator bromophenol blue enters the concentrated gel and forms a straight line, change to constant voltage electrophoresis at 150V. During the electrophoresis, observe the position of protein electrophoresis through the marker. When the indicator bromophenol blue runs to the bottom of the gel, the electrophoresis can be stopped and the transfer is ready. Cut the 0.45μm PVDF membrane into a suitable size and put it in an appropriate amount of methanol solution to activate it for more than 20s. Assemble the "sandwich" transfer structure from the negative electrode to the positive electrode (the order is black frame, sponge, filter paper, gel, PVDF membrane, filter paper, sponge, white frame), and then put the transfer clip into the transfer tank corresponding to the positive and negative electrodes. Add fast transfer solution and ice box, use 100V constant voltage for transfer, and the transfer time depends on the molecular weight of the target protein. After the transfer, wash the membrane with 1TBST 3 times, 10 minutes each time. Then place the membrane in 5% skim milk for blocking at room temperature for 1 hour. Wash the membrane with 1×TBST solution 3 times, 10 minutes each time. Incubate the primary antibody at 4℃ overnight, and dilute the antibody according to the concentration specified in the instructions. On the second day, wash the membrane with 1×TBST solution 3 times, 10 minutes each time. Incubate the secondary antibody and incubate on a shaker at room temperature for 1 hour. Wash the membrane with 1×TBST solution 3 times, 10 minutes each time. Prepare ECL working solution, add it evenly on the membrane, and develop it on the imager.

[0068] 10.ELISA test

[0069] The designated cell culture supernatants were collected for ELISA analysis. The cells were centrifuged at 300 g for 10 min to remove the cell pellet. TNC (Cloud-Clone Corp, SEB975Hu), FLNC (Cloud-Clone Corp, SEE932Hu), COL1A1 (Cloud-Clone Corp, SEA350Hu) and FN1 (Proteintech, KE00039) ELISA kits were used to detect the secretion levels of TNC, FLNC, COL1A1 and FN1 in the supernatant. The above cell experiment results are shown in Figure 4 , indicating that the expression of TNC, FLNC and collagen-related proteins COL1A1 and FN1 were upregulated after HCMECs were co-cultured with drug-resistant cells or stimulated with chemotherapeutic drugs.

[0070] 11.TIMER2.0 database was used to analyze the correlation between TNC or FLNC and CAFs infiltration, COL1A1, and FN1 expression. Figure 3 , showing that TNC and FLNC were significantly positively correlated with CAFs infiltration, COL1A1, and FN1 expressions.

[0071] Example 3

[0072] In view of the potential role of FLNC and TNC in EndMT and ECM formation, the sequence information of FLNC and TNC proteins was first obtained from the Uniprot database and imported into the Discovery Studio software for homologous sequence alignment and homology similarity calculation. The homology calculation results exported by the Discovery Studio software show that the two proteins have a certain degree of homology, and the identity value calculated by the software can reach 14.7%; at the same time, the observation of the two protein structures shows that the two proteins are both rich in β-sheet secondary structures and actin-like structures connected by Coil, so it can be considered that they have a certain degree of homology, and subsequent virtual screening can be carried out in a targeted manner.

[0073] The full-length sequences of the two proteins were obtained from the Uniprot database, and the structures of the two proteins were modeled and predicted using the AlphaFold3 software, and energy optimization was performed using Rosetta Relax. The obtained structures were used for active cavity detection and subsequent virtual docking screening. The active sites of FLNC protein and TNC protein were predicted using Sitemap software, and the two proteins were aligned and compared. The protein active site prediction results were exported from Sitemap. Analysis and comparison of the predicted binding cavities of the two proteins showed that the Sitemap output results both contained binding cavities composed of the multi-β-sheet structures of the two proteins. It is believed that the active sites of the two proteins have a high degree of homology and similarity, and subsequent virtual screening can be performed.

[0074] Pymol was used to prepare the protein structure, including dehydration and hydrogenation. The small molecule database screened was the TCMSP database of active small molecules in traditional Chinese medicine (https: / / www.tcmsp-e.com / # / database). AutodockVina was used to complete the docking. The docking box and grid parameters were set according to the way of covering the active cavity, and the sites were fully covered to find the best binding site and the best conformation of the small molecule in the active cavity. The specific docking box parameters are set as follows: the box parameters of FLNC protein: center_x = 1.596, center_y = -16.151, center_z = 44.464, size_x = 30, size_y = 34, size_z = 32, spacing = 0.581; the box parameters of TNC protein: center_x = -3.673, center_y = 36.207, center_z = -20.521, size_x = 30, size_y = 26, size_z = 20, spacing = 0.631. After the docking is completed, Vina is used to assist Python in sorting the predicted small molecules based on the binding energy predicted by the empirical force field, and the relative position of the small molecules relative to the protein and the force analysis are analyzed and plotted. The force analysis of the ligand and receptor is completed by PLIP software analysis, and the 3D conformation of the ligand-receptor is displayed using Pymol software, see Figure 5 .Depend on Figure 5 It can be seen that among the TOP3 small molecules screened, PNC has a significant inhibitory effect on FLNC protein, TNC protein, EndMT-related protein, multidrug resistance-related membrane protein ABCB1, and collagen-related protein expression FN1 / COL1A1.

[0075] Example 4

[0076] Subcutaneous tumor xenografts. 4-week-old male BALB / c nude mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. (Jiangsu) and housed under specific pathogen-free conditions. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. 3 The TMZ-R tissue blocks of different sizes (obtained before the experiment) were inoculated subcutaneously in the right axilla of balb / c nude mice. After the subcutaneous tumor was formed, the volume was increased to 100-150 mm. 3Mice were randomly treated with oral saline, TMZ (20 mg / kg), PNC (40 mg / kg), or TMZ and PNC combination therapy, and the drugs were administered daily. The body weight and tumor volume of mice were measured every 2 to 3 days after administration, and the anti-tumor effect of the compound was dynamically observed. After 10 days of administration, the mice were killed, and the tumors were surgically removed and weighed. The tumor growth curve was drawn, and the tumors were taken and photographed. The results are shown in Figure 6 ,Depend on Figure 6 It can be seen that there was no tumor reduction after TMZ administration alone, while the combined administration of TMZ and PNC significantly inhibited tumor growth and increased the sensitivity of drug-resistant cells to TMZ.

[0077] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of pomegranate peel tannin in the preparation of anti-tumor drugs.

2. The use according to claim 1, characterized in that: The anti-tumor drugs include anti-tumor drugs that improve chemotherapy sensitivity.

3. Application of pomegranate peel tannin combined with chemotherapy drugs in the preparation of anti-tumor drugs.

4. The use according to claim 3, characterized in that: The chemotherapy drugs include temozolomide or etoposide.

5. The use according to any one of claims 1 to 4, characterized in that: The tumor includes a glioma or a glioblastoma.

6. Use of pomegranate peel tannin in the preparation of inhibitors of tumor protein TNC and / or tumor protein FLNC; the amino acid sequence of TNC is shown in SEQ ID NO.1; the amino acid sequence of FLNC is shown in SEQ ID NO.

2.

7. Application of pomegranate peel tannin in the preparation of endothelial-mesenchymal transition inhibitors.

8. Use of pomegranate peel tannin in the preparation of protein expression inhibitors; the proteins include one or more of endothelial-mesenchymal transition-related proteins, multidrug resistance-related membrane proteins and collagen-related proteins.

9. An anti-tumor drug comprising pomegranate peel tannin and a chemotherapy drug.

10. The anti-tumor drug according to claim 9, characterized in that: The chemotherapy drugs include temozolomide or etoposide.

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