Application of luteolin derivative in preparation of anti-cancer drugs

Abstract

The invention discloses an application of a luteolin derivative in preparation of an anti-cancer drug, the cancer is any one of colorectal cancer, liver cancer, gastric cancer or lung cancer, the proliferation inhibition activity of LTD2 on colorectal cancer cells is superior to that of other tumor cells, network pharmacological analysis reveals that the anti-colorectal cancer core targets of LTD2 comprise AKT1, EGFR and the like, and the luteolin derivative can be used for preparing the anti-cancer drug. And the molecular docking verifies that the molecular docking has good binding capacity with a key target spot. According to the present invention, the significant effects of tumor cell growth inhibition, cell apoptosis induction and the like are provided, the anti-tumor activity is enhanced through the multi-target point synergistic effect, and the excellent safety and the strong anti-tumor effect provide the solid foundation for the subsequent preclinical research and development; the medicine is expected to become a novel candidate medicine for treating colorectal cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of a luteolin derivative in the preparation of an anti-cancer drug. Background Art

[0002] Colorectal Cancer (CRC) is one of the malignant tumors with relatively high incidence and mortality rates globally. According to the latest statistical data, the incidence rate of CRC ranks among the top in global cancers, and its mortality rate also remains high. Although traditional treatment methods such as surgery, chemotherapy, and radiotherapy can control the development of the disease to a certain extent, these methods are often accompanied by severe side effects, and patients are prone to develop drug resistance. For example, chemotherapy drugs often cause discomfort reactions such as nausea, vomiting, and hair loss, and long-term use may also lead to serious complications such as bone marrow suppression. Therefore, it is particularly urgent to develop new anti-tumor drugs with high efficiency and low toxicity.

[0003] The natural compound luteolin (Lut), as a flavonoid compound widely present in nature, has received extensive attention due to its significant anti-inflammatory, antioxidant, and potential anti-tumor activities. Research shows that luteolin not only has a wide range of biological effects, including inhibiting the proliferation of different types of tumor cells, inducing apoptosis, and regulating immune responses, but also shows potential therapeutic value in various cancer models.

[0004] However, despite the many advantages of luteolin mentioned above, the problems of its low water solubility and bioavailability have become the main obstacles restricting its wide clinical application. Due to the poor solubility of luteolin in water, its oral absorption efficiency is low, which in turn affects its in vivo bioavailability. To overcome this limitation, scientists have carried out a large amount of research work, attempting to improve the physicochemical properties of luteolin and enhance its bioavailability through chemical modification methods, with a view to developing more effective anti-cancer drugs. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of a luteolin derivative in the preparation of an anti-cancer drug, which not only shows significant efficacy in inhibiting tumor cell growth, inducing apoptosis, etc., but also has great advantages in terms of safety.

[0006] The above technical objective of the present invention is achieved through the following technical solutions:

[0007] The application of a luteolin derivative in the preparation of an anti-cancer drug, and the structural formula of the luteolin derivative is:

[0008]

[0009] Further preferably, the cancer targeted by the anti-cancer drug is any one of colorectal cancer, liver cancer, gastric cancer or lung cancer.

[0010] Further preferably, the anti-cancer drug is a targeted drug.

[0011] In summary, the present invention has the following beneficial effects:

[0012] First, the luteolin derivative LTD2 of the present invention exhibits significant efficacy in the field of anti-colorectal cancer, and its mechanism of action mainly focuses on the PI3K-AKT signaling pathway. Research shows that LTD2 has stronger anti-tumor activity compared to the parent compound luteolin. Through a series of experiments, it is found that LTD2 affects colorectal cancer cells in a concentration- and time-dependent manner, and it can effectively reduce cell viability, and the IC 50 value is in the range of 8-17 μM. In terms of cell colony formation ability, LTD2 shows obvious inhibitory effects, and can arrest the cell cycle at the G2 phase, while down-regulating the expression of CDK2.

[0013] Second, mechanism studies show that LTD2 regulates the phosphorylation level and active expression of key effector molecules such as AKT1 and NFKB1 by targeting them, thereby inhibiting the core regulatory nodes of the PI3K-AKT signaling pathway at multiple levels. At the same time, LTD2 can activate the Caspase 3-dependent apoptosis pathway, promoting cancer cell apoptosis. With the help of network pharmacology and molecular docking technology, it is further confirmed that LTD2 can play a specific role through the differential target NFKB1.

[0014] Third, in terms of safety evaluation, the results of the oral acute toxicity test on mice show that the LD 50 of LTD2 > 5 g / kg. According to the toxicity grading standard, it belongs to a practically non-toxic compound, showing good safety. Description of the Drawings

[0015] Figure 1 is the structural diagram of the luteolin derivative (LTD2);

[0016] Figure 2 shows the effects of Lut and LTD2 on the viability of HCT116 and SW480 cells at different times;

[0017] Figure 3 shows the effects of Lut and LTD2 on the colony formation ability of HCT116 cells;

[0018] Figure 4 shows the effects of Lut and LTD2 on the colony formation ability of SW480 cells;

[0019] Figure 5The effects of Lut and LTD2 on the cell cycle of HCT116 cells;

[0020] Figure 6 The effects of Lut and LTD2 on the cell cycle of SW480 cells;

[0021] Figure 7 The Venn diagram of Lut, Lut and CRC targets;

[0022] Figure 8 The molecular docking model diagrams of Lut and LTD2 with key targets;

[0023] Figure 9 The effects of Lut and LTD2 on the p-AKT and NFKB1 proteins in HCT116 cells;

[0024] Figure 10 The effects of Lut and LTD2 on the expression of p-AKT and NFKB1 proteins in HCT116 cells;

[0025] Figure 11 The effects on the body weight of female ICR mice after administration of Lut and LTD2;

[0026] Figure 12 The organ weights of female ICR mice after administration of Lut and LTD2;

[0027] Figure 13 The organ index diagrams of female ICR mice after administration of Lut and LTD2. Detailed implementation manners

[0028] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0029] Example 1, Inhibitory effect of luteolin derivative LTD2 on colorectal cancer cells

[0030] 1 Experimental materials

[0031] 1.1 Test drugs

[0032] 1.1.1 Basic information of test drugs

[0033] (1) Luteolin (Lut) with a molecular weight of 286.24 was purchased from Ron Reagent Company, batch number RH562456.

[0034] (2) Luteolin derivative (LTD2) has a molecular weight of 393.82 and batch number: SL-P1223-7, provided by Yunnan Suli Biomedical Technology Co., Ltd.

[0035] In the early stage of our laboratory, the research group of Teacher Yang Weimin obtained a class of multi-target compounds, 3'-aminoalkoxy-luteolin derivatives and their pharmaceutically acceptable salts, through chemical synthesis method, and has obtained a patent (CN201811575228.8). Among them, luteolin derivative (LTD2) was obtained by introducing -CH2CH2-N-(CH3)2 group on the 3'-phenolic hydroxyl group. Compared with luteolin, it has good water solubility, and its structure is as Figure 1 .

[0036] (3) Oxaliplatin (Oxa) has a molecular weight of 397.30, purchased from Qilu Pharmaceutical Co., Ltd. Hainan Branch, batch number AA2E0006A.

[0037] 1.1.2 Preparation of test drugs

[0038] (1) Weigh 14.31 mg of luteolin powder, add 1 mL of DMSO to dissolve it, and obtain a stock solution with a final concentration of 100 mM. Aliquot and store at -80 °C.

[0039] (2) Weigh 19.691 mg of luteolin derivative powder, add 500 μL of DMSO to dissolve it, and obtain a stock solution with a final concentration of 100 mM. Aliquot and store at -80 °C.

[0040] (3) Weigh 12 mg of oxaliplatin powder, add 1.5 mL of ultrapure water to dissolve it, and obtain a stock solution with a final concentration of 20 mM. Aliquot and store at -80 °C.

[0041] 1.2 Cell lines

[0042]

[0043] 2 Experimental methods

[0044] 2.1 Effect of luteolin derivative LTD2 on the proliferation of tumor cells such as digestive tract

[0045] (1) After digesting and counting the cells, inoculate them into a 96-well plate at 0.5x10 4 cells / well, 100 μL / well, and place them in an incubator at 37 °C, 5% CO2 saturated humidity for 24 h to allow them to adhere.

[0046] (2) Aspirate the medium in the 96-well plate. Add 100 μL / well of the medium containing different doses of the drug to the treatment group, set 5 replicates for each concentration, and add an equal volume of medium to the negative control group. Then continue to culture in the incubator for 24 h.

[0047] (3) Remove the 96-well plate from the incubator, aspirate the liquid with a pipette, wash once with 100 μL / well of PBS, aspirate the PBS, add MTT, and place in the incubator for 4 h.

[0048] (4) Aspirate the liquid in the well plate and add 150 μL / well of DMSO.

[0049] (5) Measure the OD value of each well in the microplate reader. Calculate the cell viability and IC 50 value.

[0050] 2.2 Effects of luteolin derivative LTD2 on the growth of colorectal cancer HCT116 and SW480 cells

[0051] 2.2.1 Effects of LTD2 on the proliferation of HCT116 and SW480 cells

[0052] The drug action time is 24, 48, and 72 h.

[0053] 2.2.2 Effects of LTD2 on the colony formation ability of HCT116 and SW480 cells

[0054] (1) Inoculate logarithmic growth phase colorectal cancer cells HCT116 (0.5×10 3 cells / mL), SW480 (1×10 3 cells / mL) at the density into a 6-well culture plate, and culture the cells overnight until they adhere to the wall.

[0055] (2) Add LTD2 at concentrations of 5.4, 9, 15, 25 μM and Lut, OXA (3 μM) to treat HCT116 cells, and add LTD2 at concentrations of 7.6, 12.6, 21, 35 μM and Lut, OXA (4 μM) to treat SW480 cells. Culture in the cell incubator for about 10 days, and change the fresh drug-containing culture medium every 3 days.

[0056] (3) Wash twice with PBS, fix with methanol for 30 min, wash once with PBS, stain with crystal violet for 10 min, wash 3 times with PBS, air dry, and take pictures.

[0057] (4) Calculate the colony formation rate with Image J.

[0058] 2.2.3 Effects of LTD2 on the cell cycle of HCT116 and SW480 cells

[0059] 2.2.3.1 Detection of the effect of LTD2 on the cell cycle by FCM

[0060] (1) HCT116 (0.75×10 5 cells / mL), SW480 (1×105 Cells (at a density of cells / mL) were seeded in a 6-well plate and incubated overnight in an incubator.

[0061] (2) HCT116 cells were treated with LTD2 at concentrations of 9, 15, and 25 μM, along with Lut and OXA at 3 μM, for 36 h. SW480 cells were treated with LTD2 at concentrations of 12.6, 21, and 35 μM, along with Lut and OXA at 8 μM, for 48 h.

[0062] (3) The culture medium was collected into a centrifuge tube. The cells were washed once with PBS, and the PBS was collected into the centrifuge tube. The cells were digested and collected into the centrifuge tube.

[0063] (4) Centrifuge at 800 rpm for 5 min. Discard the supernatant, wash the cells with PBS, repeat 2 times, and then centrifuge.

[0064] (5) After resuspending the cells with 250 μL of PBS, add 750 μL of absolute ethanol and incubate overnight at 4°C (to fix the cells).

[0065] (6) Centrifuge, discard the supernatant, wash once with PBS, and discard the supernatant.

[0066] (7) Resuspend the cells with 500 μL of a liquid containing propidium iodide and RNase, and incubate in a 37°C water bath for 30 min.

[0067] (8) Detect using a flow cytometer.

[0068] 2.3 Statistical analysis

[0069] The experimental data of each group were expressed as mean ± standard deviation ( ). Data analysis was performed using SPSS 22.0. For comparisons between groups that conformed to a normal distribution and homogeneous variance, paired t-tests or one-way ANOVA were used. According to α = 0.05, P < 0.05 was considered statistically significant.

[0070] 3 Experimental results

[0071] 3.1 Effects of luteolin derivative LTD2 on the proliferation of tumor cells such as those in the digestive tract

[0072] Seven human tumor cell lines from different sources (including colorectal cancer, liver cancer, gastric cancer, and lung cancer) were selected. Using Lut as a reference, the MTT method was used to evaluate the effects of LTD2 on the proliferation of tumor cells after treatment at different concentrations for 24 h. The results showed that in the same cell line, the half-maximal inhibitory concentration (IC 50 ) of LTD2 was significantly lower than that of Lut (P < 0.05), indicating that it had stronger anti-proliferative activity. At the same time, the inhibitory activity of LTD2 on the proliferation of colorectal cancer cells was better than that of other tumor cells, and its IC 50The values were distributed in the range of 8 - 17 μM, while the IC 50 of other tumor cells were all higher than 30 μM, as shown in Table 1.

[0073] Table 1 IC 50 values (μM) of LTD2 and Lut in inhibiting tumor cell proliferation ( n = 3)

[0074]

[0075] Note: * P < 0.05, ** P < 0.01, *** P < 0.001 compared with Lut.

[0076] 3.2 Effects of luteolin derivative LTD2 on the growth of colorectal cancer HCT116 and SW480 cells

[0077] 3.2.1 Effects of LTD2 on the proliferation of HCT116 and SW480 cells

[0078] The effects of the compound on the proliferation of two colorectal cancer cell lines, HCT116 and SW480, at different action times (24, 48, 72 h) were detected by the MTT method. The results showed that LTD2 could significantly inhibit the viability of the two cell lines, and its anti - proliferative effect showed concentration - and time - dependence. At the same action time, the IC 50 values of LTD2 were significantly lower than those of the Lut group (P < 0.01), as shown in Table 2. Figure 2 .

[0079] Table 2 IC 50 values (μM) of Lut and LTD2 in inhibiting the proliferation of human colorectal cancer cells at different times ( n = 3)

[0080]

[0081] 3.2.2 Effects of LTD2 on the colony - forming ability of HCT116 and SW480 cells

[0082] In HCT116 cells, compared with the control group, the positive drug OXA at 3 μM could completely inhibit the colony - forming ability of cells (P < 0.001). With the increase of the administration concentration of LTD2, the colony - forming ability of cells decreased significantly (P < 0.001). When the concentration ≥ 9 μM, it could completely inhibit the colony - forming of HCT116 cells (P < 0.001). At the same concentration (5.4, 9, 15, 25 μM), LTD2 inhibited the colony - forming ability of HCT116 cells significantly better than Lut (P < 0.05), as shown in Figure 3 .

[0083] In SW480 cells, compared with the control group, the positive drug OXA at 4 μM could completely inhibit cell colony formation (P < 0.001). With the increase in the administration concentration of LTD2, the cell colony formation ability was significantly reduced (P < 0.001). When the concentration ≥ 12.6 μM, the colony formation ability of SW480 cells could be completely inhibited (P < 0.001). At the same concentration (7.6, 12.6, 21, 35 μM), LTD2 was significantly superior to Lut in inhibiting the colony formation ability of SW480 cells (P < 0.01). See details in Figure 4 .

[0084] The above results indicate that LTD2 inhibits the colony formation ability of rectal cancer cells in a concentration-dependent manner, and its effect intensity is significantly superior to Lut at the same concentration (P < 0.05).

[0085] 3.2.3 Effects of LTD2 on the cell cycle of HCT116 and SW480 cells

[0086] 3.2.3.1 Effects of LTD2 on the cell cycle of HCT116 and SW480 cells

[0087] In HCT116 cells, the cells in G1 phase, S phase and G2 phase in the control group were 42.90 ± 1.76%, 39.6 ± 1.47%, 16.02 ± 1.38% respectively; after treatment with 3 μM OXA for 36 h, the proportion of G2 phase increased to 42.46 ± 1.49% (compared with the control group, P < 0.001). When treated with LTD2, at the concentrations of 9 μM and 15 μM for 36 h, the proportions of G2 phase were 25.28 ± 3.26% and 33.72 ± 3.26% respectively (compared with the control group, P < 0.05). When Lut was used at the concentrations of 9, 15, 25 μM for 36 h, the proportions in G2 phase were 15.18 ± 4.8%, 29.6 ± 3.86%, 39.7 ± 1.28% respectively. When the concentration of LTD2 reached 25 μM, the proportion of G1 phase increased significantly to 75.23 ± 1.91% (compared with the control group, P < 0.001) (see details in Table 3, Figure 5 ).

[0088] In the SW480 cells, the percentages of cells in the G1, S, and G2 phases in the control group were 55.8 ± 10.83%, 34.69 ± 7.86%, and 7.82 ± 4.45%, respectively. After treatment with 8 μM OXA for 48 h, the proportion of cells in the G2 phase increased significantly to 21.78 ± 7.93% (P < 0.01 compared with the control group). After treatment with LTD2 at concentrations of 12.6, 21, and 35 μM for 48 h, the proportions of cells in the G2 phase were 10.47 ± 3.56%, 29.37 ± 6.01%, and 37.13 ± 2.76%, respectively, and those at 21 μM and 35 μM were significant (P < 0.01 compared with the control group). After treatment with Lut at concentrations of 12.6, 21, and 35 μM for 48 h, the percentages of cells in the G2 phase were 9.27 ± 2.62%, 12.95 ± 4.21%, and 20.08 ± 1.03%, respectively. (See Table 4, Figure 6 ).

[0089] The above results indicate that OXA, LTD2, and Lut arrest HCT116 and SW480 cells in the G2 phase. In addition, at the highest dose, LTD2 arrests HCT116 cells in the G1 phase.

[0090] Table 3 Effects of Lut and LTD2 on the cell cycle of HCT116 cells ( n = 3)

[0091]

[0092] Note: ** P < 0.01, *** P < 0.001 compared with Control.

[0093] Table 4 Effects of Lut and LTD2 on the cell cycle of SW480 cells ( n = 3)

[0094]

[0095] Note: ** P < 0.01, *** P < 0.001 compared with Control.

[0096] Example 2 Exploration of the mechanism of action of luteolin derivative LTD2 against colorectal cancer

[0097] 1 Experimental methods

[0098] 1.1 Network pharmacology research

[0099] 1.1.1 Screening of drug targets

[0100] Download the structural formula and SMILES number of Lut from the PubChem database, then draw the structural formula of LTD2 based on luteolin, import the structural formula into the SwissTargetPrediction and Superpred databases to predict related targets and remove duplicates, and convert the obtained Uniprot ID to gene names through ID mapping in the Uniprot database.

[0101] 1.1.2 Screening of colorectal cancer disease targets

[0102] Search the 4 databases of GeneCards, Drugbank, OMIM, and TTD with the search term "Colorectal Cancer" to search for targets related to CRC and merge the targets obtained from each database. The species setting of the GeneCards database is "Homo sapiens", and for the obtained data, select genes with a "Relevance score" > 1.0; use the data in the "Approved Symbol" column of the OMIM database as targets; retain all data in the Drugbank database; click Target NetInfo in the TTD database in turn to view genes and summarize. Finally, summarize and remove duplicates to obtain disease target information.

[0103] 1.1.3 Screening of drug-disease common targets

[0104] Import the LTD2 and Lut targets and CRC-related targets into the Venny 2.1.0 online analysis platform to obtain a Venn diagram and analyze the identical and different genes between the two.

[0105] 1.1.4 Drug-disease target enrichment analysis

[0106] Import the common targets of LTD2, Lut and CRC into the DAVID database, set the biological species as "HomoSapiens", and perform GO and KEGG signaling pathway enrichment analysis. The results are presented in the form of bar charts, bubble charts, etc., and explore the possible mechanism of action of LTD2 compared with Lut against CRC according to the enrichment degree of key pathways.

[0107] 1.1.5 Drug-disease protein interaction network and screening of key targets

[0108] The target was imported into the STRING platform, with the confidence level set at 0.4, and the PPI results were obtained and the data was downloaded. The PPI data was imported into the Cytoscape 3.6.0 software for visualization and the interaction node scores were obtained. It was calculated using the "MCC, Degree, MNC, Closeness, EPC, Radiality, Betweenness, Stress" algorithms in the CytoHubba plugin. The top 10 scores were used as the key genes for each algorithm. Finally, the key targets identified by the above 8 different algorithms were intersected, and the intersected targets were defined as "key targets".

[0109] 1.2 Molecular docking

[0110] (1) From the PubChem database, the 2D structure diagram of Lut was downloaded and saved in the sdf format. The structure of LTD2 was drawn in ChemDraw 20.0 and exported as an sdf structure.

[0111] (2) The sdf structure of the drug was imported into the Chem 3D software, energy minimization was performed, and it was exported in the pdb format.

[0112] (3) The Uniprot ID of the key targets in 3.1.5 above was selected and imported into the pdb structure downloaded from the PDB database.

[0113] (4) Molecular docking was performed using the AutoDockTools software.

[0114] (5) The docking model was visualized through the Pymol software.

[0115] 1.3 Experimental verification of core targets and differential target proteins

[0116] According to the key signaling pathways enriched by KEGG in 3.1.4 above and combined with the key targets in 3.1.5, the corresponding target proteins were screened out, and the expression changes of related proteins were detected by Western Blot.

[0117] 2 Research results

[0118] 2.1 Network pharmacology research

[0119] 2.1.1 Screening of drug targets

[0120] The relevant targets predicted by the SwissTargetPrediction and Superpred databases were de-duplicated, and finally 200 targets of Lut and 201 targets of LTD2 were screened out.

[0121] 2.1.2 Screening of colorectal cancer disease targets

[0122] 13,029 targets were screened out through the GeneCards database. According to the median method, 1,130 targets were screened out. 168 targets were screened out from OMIM, and 100 targets were screened out from the TTD database. After removing duplicates, 1,456 CRC targets were screened out.

[0123] 2.1.3 Screening of drug-disease common targets

[0124] There were 82 potential common targets after the intersection of Lut drug targets and CRC disease targets, and there were also 82 for LTD2. Among them, there were 64 common targets between Lut, LTD2 and CRC, and 18 intersection targets with CRC for each. See Figure 7 . The 18 targets different between LTD2 and Lut were PLA2G2A, KIT, CHEK2, CHEK1, INSR, PIK3CG, NOS2, PTPN1, PTK2B, MDM4, PPARD, NFKB1, NFE2L2, KEAP1, HPRT1, HDAC9, FOLH1, ADAM10.

[0125] 2.2 Molecular docking

[0126] Molecular docking of Lut and LTD2 with key targets in the PPI network was performed using AutoDock Vina software. The results showed that the binding free energy (Affinity) of both with all targets was significantly lower than -5 kcal / mol (Table 5), indicating that Lut and LTD2 can not only bind to core targets efficiently, but also show considerable binding ability to these targets. To more intuitively display these binding modes, we used PyMol software for molecular docking visualization ( Figure 8 ), which detailed the interaction between the receptor and the ligand.

[0127] Table 5 Molecular docking score results of Lut, LTD2 and core targets

[0128]

[0129]

[0130] 2.3 Experimental verification of core targets and differential target proteins

[0131] In HCT116 cells, OXA treatment significantly inhibited the expression of p-AKT and NFKB1 proteins (P<0.01 compared with the control group). With the increase in the concentrations of LTD2 and Lut, the expression of p-AKT protein was significantly downregulated (P<0.01 compared with the control group). At the same concentrations (9 μM and 15 μM), the inhibitory effect of LTD2 on p-AKT was significantly stronger than that of Lut( Figure 9 A, B). LTD2 treatment significantly reduced the expression of NFKB1 (P<0.01 compared with the control group), and the effect was enhanced with the increase in concentration. After Lut treatment, there was no obvious change trend in NFKB1 protein (P>0.05 compared with the control group). At the same concentrations (9 μM and 15 μM), the effect of LTD2 on the downregulation of NFKB1 protein was stronger than that of Lut (P<0.01)( Figure 9 A, C).

[0132] In SW480 cells, OXA treatment significantly inhibited the expression of p-AKT and NFKB1 proteins (P<0.05 compared with the control group). With the increase in the concentration of LTD2, the expression of p-AKT protein was significantly downregulated (P<0.01 compared with the control group). At the same concentrations (12.6 μM and 21 μM), the effect of LTD2 on the downregulation of p-AKT protein was stronger than that of Lut (P<0.05)( Figure 10 A, B). With the increase in the concentration of LTD2, the expression of NFKB1 protein was significantly downregulated (P<0.05 compared with the control group). After Lut treatment, there was no obvious change trend in NFKB1 protein (P>0.05 compared with the control group). At the same concentrations (12.6 μM and 21 μM), the effect of LTD2 on the downregulation of NFKB1 protein was stronger than that of Lut (P<0.05)( Figure 10 A, C).

[0133] The above results indicate that LTD2 can effectively inhibit the expression of p-AKT and NFKB1 proteins in HCT116 and SW480 cells, and its inhibitory effect is stronger than that of Lut.

[0134] Example 3, Oral Acute Toxicity Study of Luteolin Derivative LTD2 in Mice 1 Experimental Method

[0135] 1.1 Preparation of Test Drugs

[0136] (1) Weigh 0.5512 g of Lut powder, add drinking water to 4.4 mL, and stir well to make it uniform, preparing a 125 mg / mL yellow suspension;

[0137] (2) Weigh 0.5514 g of LTD2 powder, add drinking water to 4.4 mL, and stir well to make it uniform, preparing a 125 mg / mL light yellow suspension.

[0138] 1.2 Grouping of experimental animals

[0139] Fifteen ICR female mice, weighing 18.0 - 22.0 g, were selected. After weighing them respectively, they were randomly grouped by body weight into a solvent control group and two drug - administered groups (Lut group, LTD2 group), with 5 mice in each group. They were stained with saturated picric acid for hair marking, and each group of animals was identified and numbered respectively.

[0140] 1.3 Administration of drugs to experimental animals

[0141] Referring to the "Technical Guidelines for Single - Dose Toxicity Studies of Drugs" and according to the results of preliminary tests, in this experiment, the maximum - dose method was used for gavage administration at a dose of 5 g / kg. The administration volume was 40 mL / kg.bw, and gavage administration was carried out once. The animals were fasted for 12 h before the experiment without water deprivation.

[0142] 1.4 Observation indicators

[0143] (1) General condition observation: During and after drug administration, the appearance signs, behavioral activities, mental status, facial features, genitalia, and fecal characteristics of the animals were observed, and the observation was continuous for 14 days.

[0144] (2) Body weight changes: The body weights were measured once on the 0th, 3rd, 7th, and 14th days of the experiment, and the changes in body weight were observed and recorded.

[0145] (3) Toxic reactions and deaths: The symptoms of toxic reactions in the animals, the onset and duration, whether the toxic reactions were reversible, the time and number of animal deaths, etc. were observed and recorded.

[0146] (4) Gross observation during systematic dissection: At the end of the experiment, the animals were subjected to systematic dissection, and whether there was abnormal fluid accumulation in the thoracic and abdominal cavities was observed macroscopically. The shape, size, color, and texture of each organ and tissue were examined. Whether there were abnormal conditions such as congestion, hemorrhage, nodules, ulcers, abscesses, adhesions, necrosis, and masses on the surface of the organs and tissues was also checked.

[0147] 1.5 Statistical analysis

[0148] SPSS 22.0 statistical software was used for analysis, and all data were expressed as mean ± standard deviation One - way ANOVA was used to statistically analyze the body weights of the animals, and P < 0.05 was considered statistically significant.

[0149] 2 Experimental results

[0150] 2.1 General conditions and signs

[0151] After intragastric administration of the test substance, there were no obvious abnormalities in the spontaneous activities, diet, and mental states of the mice in the vehicle control group, LTD2 group, and Lut group. After continuous observation for 14 days, all the experimental animals survived. It is indicated that the LD 50 > 5 g / kg for Lut and LTD2.

[0152] 2.2 Body weight changes

[0153] During the test period, the body weights of the animals in each group increased. Except that on the 3rd day, the body weight of the LTD2 group was slightly lower than that of the vehicle control group, and there was a significant difference in statistical comparison (P < 0.05), there were no significant differences in body weights among the groups on the other days (P > 0.05). There was also no significant difference in the weight gain of the animals at the end of the experiment among the groups (P > 0.05), as shown in Table 6, Figure 11 .

[0154] Table 6 Effects of Lut and LTD2 on the body weight of female ICR mice (g) ( n = 5)

[0155]

[0156]

[0157] Note: * P < 0.05, compared with the vehicle group;

[0158] 2.3 Gross anatomical observation

[0159] (1) Gross anatomical observation was performed on all the surviving experimental animals. The textures and colors of the main organs, including the heart, liver, spleen, lungs, kidneys, and thymus, were normal, and no obvious abnormal changes such as enlargement, adhesion, and congestion were observed.

[0160] (2) When comparing the weights of the organs among the groups, except that the weight of the lungs in the Lut group was slightly higher than that of the vehicle control group, and there was a significant difference in statistical comparison (P < 0.05), there were no significant differences in the weights of the other organs among the groups (P > 0.05), as shown in Table 7, Figure 12 ; and there were also no significant differences in the organ coefficients among the groups (P > 0.05), as shown in Table 8, Figure 13 .

[0161] Table 7 Effects of Lut and LTD2 on the organ weights of female ICR mice (g) ( n = 5)

[0162]

[0163] Note: * P < 0.05, compared with the vehicle group.

[0164] Table 8 Effects of Lut and LTD2 on the organ coefficients of female ICR mice (%) ( n = 5)

[0165]

[0166] Note: P > 0.05, compared with the solvent group.

[0167] By gavage administration of 5 g / kg of luteolin and its derivatives to mice and observing them for the subsequent 14 days, the results showed that there were no obvious abnormalities in the spontaneous activity, diet, and mental state of the mice in the solvent control group, Lut group, and LTD2 group. The body weights of the mice all showed a stable growth trend, and their growth was not inhibited, nor was there any obvious organ toxicity or organic damage induced. All animals survived during the 14-day observation period, suggesting that the LD 50 of both LTD2 and Lut is greater than 5 g / kg, and they are classified as practically non-toxic compounds according to the OECD acute toxicity classification standard. This provides preliminary but important safety data support for subsequent research. These multi-dimensional evidences indicate that based on animal experiment and clinical trial data, luteolin exhibits reliable safety characteristics and broad application prospects throughout the whole process from basic research to clinical translation.

[0168] The above embodiments are only explanations of the present invention and not limitations thereof. Those skilled in the art can make modifications to the embodiments without creative contributions according to their needs after reading this specification, but as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. Use of a luteolin derivative in the preparation of an anti-cancer drug, characterized in that, The structural formula of the luteolin derivative is as follows:

2. Use of a luteolin derivative according to claim 1 in the preparation of an anti-cancer drug, characterized in that: The cancer targeted by the anti-cancer drug is any one of colorectal cancer, liver cancer, gastric cancer or lung cancer.

3. Use of a luteolin derivative according to claim 1 in the preparation of an anti-cancer drug, characterized in that: The anti-cancer drug is a targeted drug.

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