Application of phenylpropanoid glycoside compound in preparation of antitumor drugs

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of phenylpropanoid glycoside compounds in preparation of antitumor drugs. The phenylpropanoid glycoside compound, namely, the rhodoside A, is derived from a secondary metabolite fermented by artemisia japonica endophytic Pestalotiopsis uvicola GMH31, can be used as a sensitizer for remarkably improving the sensitivity of an anti-tumor drug to multidrug-resistant tumor cells, and can be used for up-regulating intracellular reactive oxygen species (ROS), down-regulating mitochondrial membrane potential (MMP) and targeting apoptosis genes Bax and Bcl-2, so that the rhodoside A can be used for treating multidrug-resistant tumor cells. Further, the level of Cytc in cytoplasm is obviously increased, the level of Caspase3 / 9 is obviously reduced, and the level of activated cleaved-Caspase3 / 9 is obviously increased; meanwhile, a P-gp efflux transport function can be inhibited, efflux of anti-tumor drugs is reduced, accumulation of the drugs in cells is increased, and therefore the sensitization effect of the anti-tumor drugs is achieved.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical technology, and specifically relates to the application of phenylpropanoid glycoside compounds in the preparation of anti-tumor drugs. Background Art

[0002] Malignant tumors are major diseases threatening human health and life, and are a common challenge faced globally. Drug treatment is one of the very important treatment means for tumors. However, with the use of drugs, tumors will gradually become insensitive to drugs and develop drug resistance during chemotherapy, reducing the drug efficacy or even making it ineffective. Multidrug Resistance (MDR) refers to the situation where when tumor cells develop resistance to one anti-tumor drug, they also develop resistance to many other anti-tumor drugs with different structures and mechanisms of action. The emergence of drug resistance has greatly reduced the expected efficacy of clinical chemotherapy drugs and is the main reason for the failure of many tumor treatments.

[0003] For a long time, scientific research workers have been searching for drugs that can overcome tumor multidrug resistance, but so far, no drugs that can reverse or overcome tumor drug resistance have been successfully applied clinically. Therefore, continuing to explore the molecular mechanism of tumor chemotherapy drug resistance, discovering new compounds that can overcome tumor drug resistance, and promoting their R & D process are still important tasks in tumor biology and tumor pharmacology research.

[0004] Phenylpropanoid glycoside compounds, also known as phenylethanoid glycoside compounds, are a class of natural glycosides containing substituted phenethyl and cinnamoyl groups, and are widely present in dicotyledonous plants. Since the first phenylpropanoid glycoside compound was isolated in 1963, the number of such compounds isolated has been increasing. With the continuous in-depth research on this class of compounds, pharmacological experiments have shown that phenylpropanoid glycoside compounds have a wide range of significant biological activities, such as antibacterial, anti-inflammatory, anti-tumor, antiviral, antioxidant, liver protection, base repair, etc. However, so far, there has been no research report on the use of phenylpropanoid glycoside compound - leucosceptoside A as a sensitizer for anti-tumor drugs and its sensitization mechanism. Summary of the Invention

[0005] To solve the above problems, this application provides the application of phenylpropanoid glycoside compounds in the preparation of anti-tumor drugs, specifically as follows:

[0006] The application of phenylpropanoid glycoside compound leucosceptoside A in the preparation of anti-tumor drugs.

[0007] The use of leucosceptoside A as a sensitizer for anti-tumor drugs, and the molecular formula of leucosceptoside A is C 30 H 38 O 15, its chemical structural formula is shown in the figure below. It can up-regulate intracellular reactive oxygen species (ROS), down-regulate mitochondrial membrane potential (MMP), target apoptosis genes Bax and Bcl-2, and then significantly increase the level of Cytc in the cytoplasm, significantly reduce the levels of Caspase3 / 9, and significantly increase the levels of activated cleaved-Caspase3 / 9. At the same time, it can also inhibit the efflux transport function of P-gp, reduce the efflux of anti-tumor drugs, increase the accumulation of drugs in cells, and thus play a role in sensitizing anti-tumor drugs.

[0008] The specific structural formula of the leucosceptoside A is as follows:

[0009]

[0010] Furthermore, the tumor is breast cancer, lung cancer, or liver cancer.

[0011] Furthermore, the preparation of the anti-tumor drug is to prepare or be used as a sensitizer for anti-tumor drugs.

[0012] Furthermore, the sensitizer for anti-tumor drugs is a sensitizer for doxorubicin and paclitaxel.

[0013] An anti-tumor drug sensitizer contains the phenylpropanoid glycoside compound leucosceptoside A.

[0014] Furthermore, the anti-tumor drug is any one or more of doxorubicin and paclitaxel.

[0015] An anti-tumor drug contains the phenylpropanoid glycoside compound leucosceptoside A and anti-tumor components. The anti-tumor components are selected from: compounds that affect RNA synthesis during tumor cell transcription, such as doxorubicin, daunorubicin, epirubicin, pirarubicin, etc.; cytotoxic compounds, such as platinum compounds including cisplatin and oxaliplatin; compounds that affect nucleic acid synthesis during the cell cycle, such as 5-fluorouracil; compounds that affect the synthesis and function of tumor cell proteins, such as vincristine, vinorelbine, paclitaxel, docetaxel; compounds that affect hormone levels, such as tamoxifen; preferably doxorubicin or paclitaxel.

[0016] Furthermore, the dosage form of the drug includes oral preparations, injections, and inhalants.

[0017] In summary, the beneficial effects of this application are as follows:

[0018] (1) Through in vitro experiments, this invention uses doxorubicin-resistant human breast cancer cells MCF-7 / ADR, and through the MTT method, it detects the inhibitory effect of a phenylpropanoid glycoside compound, leucosceptoside A, on the proliferation of MCF-7 / ADR cells and its cytotoxicity to human normal liver cells LO2 when used alone or in combination with doxorubicin / paclitaxel. Then, through experiments such as rhodamine 123 intracellular accumulation, intracellular reactive oxygen species level and mitochondrial membrane potential measurement, and WB, it is found that leucosceptoside A mainly upregulates intracellular reactive oxygen species (ROS), downregulates mitochondrial membrane potential (MMP), targets apoptosis genes Bax and Bcl-2, thereby significantly increasing the level of Cytc in the cytoplasm, significantly decreasing the levels of Caspase3 / 9, and significantly increasing the level of activated cleaved-Caspase3 / 9; at the same time, it can also inhibit the efflux transport function of P-gp, reduce the efflux of anti-tumor drugs, and increase the accumulation of drugs in cells, thus playing a role in sensitizing anti-tumor drugs. Based on the above experimental results, it is shown that leucosceptoside A can be used as a sensitizer for anti-tumor drugs.

[0019] (2) This invention uses the MTT method to detect the effects of leucosceptoside A alone or in combination with doxorubicin / paclitaxel on the survival rates of A549 / Taxol cells and HepG2 / ADR cells, in order to study whether leucosceptoside A still has an inhibitory effect on cell proliferation in A549 / Taxol cells and HepG2 / ADR cells after drug resistance occurs. The results show that compared with the positive drug verapamil, leucosceptoside A has lower cytotoxicity and higher safety to human normal liver cells LO2; and when leucosceptoside A is used alone in A549 / Taxol cells and HepG2 / ADR cells, it has almost no anti-tumor activity. When 10 μM leucosceptoside A is used in combination with doxorubicin and paclitaxel respectively, the proliferation of drug-resistant cells is significantly inhibited, and the reversal multiples are higher or comparable to the positive drug verapamil, indicating that leucosceptoside A can increase the sensitivity of doxorubicin and paclitaxel to different types of drug-resistant tumor cells.

[0020] (3) The phenylpropanoid glycoside compound of this invention, leucosceptoside A, is derived from the secondary metabolite fermented by the endophytic fungus Pestalotiopsis uvicola GMH31 of Artemisia japonica Thunb., and is a high-content component. It can be fermented on a large scale, at low cost, and without pollution, and high-yield leucosceptoside A can be obtained by conventional means. This invention discloses for the first time the use of a phenylpropanoid glycoside compound as an anti-tumor drug sensitizer and its mechanism of action, which not only expands the use of this compound, but also provides a new direction for the research and development of new anti-tumor drug sensitizers. The phenylpropanoid glycoside compound of this invention, leucosceptoside A, as an anti-tumor drug sensitizer, has significant effects, small toxic and side effects, and clear action targets.

[0021] (4) Aiming at the problem of multi-drug resistance in the process of treating tumors with anti-tumor drugs, the present invention provides the use of a phenylpropanoid glycoside compound, leucosceptoside A, as an anti-tumor drug sensitizer and its mechanism of action, providing a new candidate molecule for the research and development of anti-tumor drug sensitizers. Description of the Drawings

[0022] Figure 1 It is a diagram showing the effect of leucosceptoside A on the accumulation of rhodamine 123 in MCF-7 / ADR cells.

[0023] Figure 2 It is a diagram showing the effect of leucosceptoside A on the level of reactive oxygen species (ROS) in MCF-7 / ADR cells.

[0024] Figure 3 It is a diagram showing the effect of leucosceptoside A on the change of mitochondrial membrane potential in MCF-7 / ADR cells.

[0025] Figure 4 It is a diagram showing the effect of leucosceptoside A on the protein expression of P-gp, Bcl-2, Bax, Cytc, Caspase-3 / 9, and cleaved-Caspase3 / 9 in MCF-7 / ADR cells.

[0026] Figure 5 It is a diagram showing the effect of leucosceptoside A on the tumor mass of each group. Detailed Embodiments

[0027] The following further elaborates on the detailed embodiments of the present application. However, the present application is not limited to these embodiments. Any improvement or substitution based on the basic spirit of this embodiment still falls within the scope protected by the claims of the present application.

[0028] Example 1: Leucosceptoside A reverses the resistance of MCF-7 / ADR cells, A549 / Taxol cells, and HepG2 / ADR cells to doxorubicin and paclitaxel, respectively.

[0029] To study whether leucosceptoside A still has an inhibitory effect on cell proliferation in MCF-7 / ADR cells, A549 / Taxol cells, and HepG2 / ADR cells after the development of drug resistance, the MTT method was used to detect the effects of leucosceptoside A alone and in combination with doxorubicin on the survival rates of MCF-7 / ADR cells, A549 / Taxol cells, and HepG2 / ADR cells, as well as the cytotoxicity to human normal liver cells LO2. The specific operation steps are as follows:

[0030] The wells of a 96-well plate were divided into a blank group, a control group, and a drug administration group (n = 3). MCF-7 / ADR cells, A549 / Taxol cells, HepG2 / ADR cells, and LO2 cells were seeded into the wells of the control group and the drug administration group at a density of 5×10 3 cells / well, and blank medium was added to the wells of the blank group. After culturing for 48 h, the cells were washed twice with 0.1 M phosphate buffer. Blank medium was added to the wells of the control group and the blank group, and the wells of the single drug administration group were incubated with medium containing different concentrations of leucosceptoside A for 48 h. The wells of the combined drug administration group were added with leucosceptoside A (final concentrations were 5 μM and 10 μM respectively) and a series of concentrations of doxorubicin solutions. After incubation, the medium was discarded, 10 μL of MTT solution was added and incubated for 1 - 4 h, and the OD value was measured using a multifunctional microplate reader and the IC 50 was calculated. The results are shown in Tables 1 - 4.

[0031] Table 1 IC 50 values of leucosceptoside A on LO2 human normal liver cells and multidrug-resistant tumor cells

[0032]

[0033] Table 2 Reversal effect of leucosceptoside A combined with doxorubicin and paclitaxel on MCF-7 / ADR

[0034]

[0035] Table 3 Reversal effect of leucosceptoside A combined with doxorubicin and paclitaxel on A549 / Taxol

[0036]

[0037] Table 4 Reversal effect of leucosceptoside A combined with doxorubicin and paclitaxel on HepG2 / ADR

[0038]

[0039] The results in Table 1 show that compared with the positive drug verapamil, leucosceptoside A has lower cytotoxicity and higher safety on human normal liver cells LO2; and when leucosceptoside A is used alone in MCF-7 / ADR cells, A549 / Taxol cells, and HepG2 / ADR cells, it has almost no anti-tumor activity; however, the results in Tables 2 - 4 show that when 10 μM leucosceptoside A is combined with doxorubicin and paclitaxel respectively, the proliferation of the three drug-resistant cells is significantly inhibited, and the reversal fold is higher or comparable to that of the positive drug verapamil, indicating that leucosceptoside A can increase the sensitivity of doxorubicin and paclitaxel to different types of drug-resistant tumor cells.

[0040] Example 2: Effect of leucosceptoside A on the P-gp efflux transport function of MCF-7 / ADR cells.

[0041] To study the effect of leucosceptoside A on the P-gp efflux pump function of MCF-7 / ADR cells, flow cytometry was used to detect the effect of leucosceptoside A on the fluorescence intensity of rhodamine 123 in MCF-7 / ADR cells, indirectly reflecting the effect of leucosceptoside A on the transport activity of P-gp on the cell membrane. The specific operation steps are as follows:

[0042] MCF-7 / ADR cells in the exponential growth phase were seeded in 6-well plates (1 mL) at a density of 6×10 5 cells / well. After culturing for 24 h, an MCF-7 / ADR blank group, an MCF-7 / ADR+Rh123 group; a 2.5 μM leucosceptoside A+Rh123 group; a 5.0 μM leucosceptoside A+Rh123 group; a 10 μM leucosceptoside A+Rh123 group; and a 5.0 μM verapamil+Rh123 group were set up. 10 μL of compound drug solutions at different concentrations were added to make the final concentrations 2.5 μM, 5.0 μM, and 10 μM. After incubating in the dark in the incubator for 1 h, Rh123 solution was added to the corresponding wells according to different groups to make the final concentration 5 μM, and incubation was continued in the dark for 1 h. Under dark conditions, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS, digested with trypsin, and after terminating the digestion, the cell suspension was collected, centrifuged on a centrifuge (850 r / min, 4 min), the supernatant was discarded, 300 μL of pre-cooled PBS was added, the cells were resuspended and then subjected to flow cytometry detection. The results are shown in Figure 1 .

[0043] Figure 1 The results show that when the concentration of leucosceptoside A is 2.5 μM, the fluorescence intensity of the fluorescent dye rhodamine 123 in the cells can be significantly increased, and the intracellular accumulation of rhodamine 123 at a concentration of 10 μM leucosceptoside A is significantly higher than that of rhodamine 123 in the verapamil group at the same dose. This result indicates that leucosceptoside A can affect the transport activity of P-gp in MCF-7 / ADR cells, thereby increasing the accumulation of rhodamine 123 in drug-resistant cells.

[0044] Example 3: Effect of leucosceptoside A on the level of reactive oxygen species (ROS) in MCF-7 / ADR cells.

[0045] To explore whether leucosceptoside A induces an oxidative stress response in MCF-7 / ADR cells, the DHE fluorescence staining method was used to detect the level of reactive oxygen species (ROS) in MCF-7 / ADR cells. The specific operation steps are as follows:

[0046] Weigh an appropriate amount of dihydroethidium (DHE) and dissolve it in DMSO to prepare a 10mM stock solution. When using, take 5μL of 10mM DHE stock solution and add it to 10mL cell culture medium. Mix well to obtain a 5μM DHE working solution. Count the MCF-7 / ADR cells and adjust the density to 3×10 cells per well. 4 The cells were embedded in a 24-well plate. After 12 hours of culture, the cells were starved for 12 hours after being attached to the wall. Then the culture medium was replaced with a culture medium containing drugs and 2% serum in a preset concentration gradient. Three replicate wells were set for each concentration. After 48 hours of culture, the liquid in the wells was discarded, and 500 μL of DHE staining working solution was added after washing 3 times with PBS. After incubation at 37°C for 20 minutes, the working solution was discarded, and 500 μL of buffer was added, and then observed and photographed under an inverted fluorescence microscope.

[0047] Figure 2 The results showed that oryzacoside A can increase the ROS level in MCF-7 / ADR cells in a dose-dependent manner, and the upregulation of ROS level by 10 μM oryzacoside A is significantly higher than that by 10 μM Capivasertib. This result indicates that oryzacoside A can induce oxidative damage in MCF-7 / ADR cells.

[0048] Example 4: Effect of scutellaria glycoside A on mitochondrial membrane potential (MMP) in MCF-7 / ADR cells.

[0049] High levels of ROS may damage mitochondria. In order to explore whether scutellaria glycoside A can damage mitochondria in MCF-7 / ADR cells, the changes in mitochondrial membrane potential (MMP) of MCF-7 / ADR cells after 24 hours of scutellaria glycoside A or Capivasertib treatment were detected to reflect the integrity of mitochondria. The specific steps are as follows:

[0050] Take 50μL Mito-Tracker Red CMXRos solution and add 420μL anhydrous DMSO. Mix thoroughly to obtain a stock solution with a concentration of 200μM. After appropriate aliquoting, store at -20℃ in the dark. When using, take 10μL of the stock solution and add it to 10mL of cell culture medium. After mixing, it will be a 200nM Mito-Tracker Red CMXRos working solution. The working solution, PBS, and culture medium need to be heated in a 37℃ water bath in advance. Count the MCF-7 / ADR cells and adjust the density to 3×10 per well. 4Cells were seeded into 24-well plates. After culturing for 12 h, when the cells adhered to the wall, the medium was changed to serum-free medium for starvation for 12 h. Subsequently, the medium was changed to a medium containing drugs at a preset concentration gradient and 2% serum. Three replicate wells were set for each concentration. After culturing for 48 h, the liquid in the wells was discarded, washed 3 times with PBS, 500 μL of working solution was added, incubated at 37 °C for 20 min, the working solution was washed away with PBS, and the culture medium was added. Immediately, observation and photography were carried out under an inverted fluorescence microscope.

[0051] Figure 3 The results showed that acteoside A down-regulated the MMP level in MCF-7 / ADR cells in a dose-dependent manner, and the effect of 10 μM acteoside A on down-regulating MMP was more obvious than that of 10 μM Capivasertib. This result indicated that acteoside A could induce mitochondrial damage in MCF-7 / ADR cells.

[0052] Example 5: Effects of acteoside A on the protein expression of P-gp, Bcl-2, Bax, Cytc, Casepase-3 / 9, and Cleaved-Casepase-3 / 9 in MCF-7 / ADR cells.

[0053] To investigate whether acteoside A could inhibit the expression of P-gp protein and whether it could induce apoptosis of MCF-7 / ADR cells through the mitochondrial pathway, protein immunoblotting was used to detect P-gp and mitochondrial apoptosis-related proteins in MCF-7 / ADR cells. The specific operation steps were as follows:

[0054] When the MCF-7 / ADR cells reached more than 80% confluence in a 10-cm round culture dish, they were digested with trypsin. After centrifugation, the cells were made into a cell suspension and subcultured into six round culture dishes. After 24 h, they were divided into six groups: blank group, 5 μM paclitaxel alone group, 2.5 μM leucosceptoside A alone group, 5 μM leucosceptoside A alone group, 10 μM leucosceptoside A alone group, and 5 μM leucosceptoside A + 5 μM paclitaxel combination group. Different concentrations of compound solutions were added, and the cells were collected after continued culture for 24 h. Protease inhibitor and phosphatase inhibitor were added to the RIPA protein lysate at a ratio of 100:1. The cells were washed three times with pre-cooled PBS, cell lysate was added, and after standing for 5 min, the cells were scraped off with a cell scraper and transferred to a 1.5-mL EP tube. After repeated pipetting, it was vortexed for 3 min and then left standing on ice for 15 min. This step (vortexing - standing) was repeated three times, and then the EP tube was centrifuged at 12,000 rpm at 4 °C for 15 min. After centrifugation, the supernatant was transferred to a new EP tube, and the protein concentration was measured according to the BCA kit instructions. The protein with the measured concentration, lysate, and loading buffer (5×) were mixed at a pre-calculated ratio so that the final concentration of all samples was 3 mg / mL. They were boiled in a water bath for 10 min to denature the protein, cooled to room temperature, and then loaded onto the gel or stored at -20 °C in the refrigerator for later use. Western blot technology was used to detect the expression levels of P-gp, Bcl-2, Bax, Cytc, Caspase-3 / 9, and Cleaved-Caspase-3 / 9.

[0055] Figure 4 The Western blot results showed that after treatment with leucosceptoside A for 48 h, the level of the pro-apoptotic gene Bax in MCF-7 / ADR cells increased, the level of the anti-apoptotic gene Bcl-2 decreased, the level of Cytc in the cytoplasm increased significantly, the level of Caspase3 / 9 decreased significantly, and the level of the activated form cleaved-Caspase3 / 9 increased significantly, but there was no effect on the expression level of P-gp. This indicates that leucosceptoside A can induce mitochondrial apoptosis in drug-resistant MCF-7 / ADR cells in a dose-dependent manner, but cannot inhibit the expression of P-gp protein.

[0056] Example 6: The sensitizing effect of leucosceptoside A on antitumor drugs in animals.

[0057] To study the sensitizing effect of leucosceptoside A on antitumor drugs in animals, a tumor-bearing mouse model was established using multi-drug resistant breast cancer cells MCF-7 / ADR cells. The MCF-7 / ADR cells were made into a single-cell suspension with PBS, and the cell concentration was adjusted to 5×10 7Nude mice, female, 5 weeks old, weighing (18±2) g, were injected with 100 μL of the above system under sterile conditions into the subnipple fat pad of the mice. When the tumor injection site formed a visible size (about 150 mm 3 ) tumor. The mice were divided into four groups: a blank group, a group that received intraperitoneal injection of 10 mg / kg paclitaxel alone, a low-dose group that received combined intragastric administration of 10 mg / kg of scutellaria glycoside A and 10 mg / kg of paclitaxel, a medium-dose group that received combined intragastric administration of 20 mg / kg of scutellaria glycoside A and 10 mg / kg of paclitaxel, a high-dose group that received combined intragastric administration of 40 mg / kg of scutellaria glycoside A and 10 mg / kg of paclitaxel, and a positive control group that received combined intragastric administration of 20 mg / kg of verapamil and 10 mg / kg of paclitaxel. The drugs were given every other day for a total of 7 times. After the experiment, the tumor tissue was peeled off and the tumor weight was measured. The results are shown in Figure 5 shown.

[0058] Figure 5 The experimental results showed that the combination of oryzacoside A and paclitaxel chemotherapy had different degrees of inhibitory effect on tumor growth in tumor-bearing mice; compared with the paclitaxel group, the group combined with oryzacoside A had enhanced tumor inhibition, and the tumor inhibition was dose-dependent with oryzacoside A; the tumor inhibition effect of the group combined with medium-dose oryzacoside A was significantly higher than that of the group combined with the same dose of verapamil, indicating that oryzacoside A combined with paclitaxel can reverse tumor multidrug resistance. In addition, during the entire administration process, the animals in each group had good tolerance to the drugs given, and the weight of the animals did not change significantly. There was 33% death in the paclitaxel combined with verapamil group, and no death occurred in the high, medium, and low dose groups of paclitaxel combined with oryzacoside A, with good tolerance and low potential toxicity.

[0059] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. Application of phenylpropanoid glycoside compound cynaroside A in the preparation of anti-tumor drugs.

2. The use according to claim 1, characterized in that The tumor is breast cancer, lung cancer, or liver cancer.

3. The use according to claim 1, characterized in that The preparation of anti-tumor drugs is to prepare or use anti-tumor drug sensitizers.

4. The use according to claim 3, characterized in that The anti-tumor drug sensitizer is a sensitizer for doxorubicin and paclitaxel.

5. The use according to claim 1, characterized in that The specific structural formula of the oryzae glycoside A is as follows:

6. An anti-tumor drug sensitizer, characterized in that: Contains the phenylpropanoid glycoside compound cynaroside A.

7. The anti-tumor drug sensitizer according to claim 6, characterized in that: The anti-tumor drug is any one or more of doxorubicin and paclitaxel.

8. An anti-tumor drug, characterized in that: Contains phenylpropanoid glycoside compound scutellariae A and anti-tumor ingredients.

9. The antitumor drug according to claim 8, characterized in that: The anti-tumor component is any one or more of doxorubicin and paclitaxel.

10. The anti-tumor drug according to claim 8, characterized in that: The dosage forms of the drug include oral preparation, injection, and inhalation.

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