Application of glehnia littoralis in preparation of medicine

Abstract

The invention relates to the technical field of application of glehnia littoralis, in particular to application of glehnia littoralis in preparation of drugs, and discloses new application of glehnia littoralis in preparation of drugs for preventing postoperative recurrence and metastasis of tumors, and the glehnia littoralis is applied to preparation of drugs for preventing postoperative recurrence and metastasis of tumors under the concentration of not causing cytotoxicity and toxic and side effects. Through multiple key steps of intervening tumor metastasis, including inhibition of adhesion of circulating tumor cells on vascular endothelial cells, inhibition of EMT transformation of the circulating tumor cells, inhibition of trans-endothelial migration of the circulating tumor cells and the like, the effect of preventing tumor metastasis or treating tumors is achieved, and meanwhile, the activity of immune cells is improved; the traditional Chinese medicine composition has the advantages of being capable of enhancing the killing ability of immune cells to circulating tumor cells, prolonging the lifetime, having extremely high safety, and solving the problems that the immune system and the rehabilitation ability are damaged in anti-cancer treatment by traditional medicines, the traditional cancer prevention and treatment strategy is limited and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of the application of glehnol, and particularly to the application of glehnol in the preparation of drugs. Background Art

[0002] Tumor metastasis is a highly complex biological process, involving changes in the morphology and gaps of in-situ cancer cells, enhanced motility, detachment from the primary site, penetration of vascular endothelium into the blood circulation to form circulating tumor cells (CTCs), evasion of immune attack in the circulatory system, adhesion and transendothelial migration in specific organs, and proliferation and angiogenesis in new organs, etc.

[0003] Currently, tumor treatment strategies cover diverse means such as radiotherapy, chemotherapy, immunotherapy, and surgical treatment, and different treatment plans are implemented according to individual disease conditions. However, these therapies are still difficult to contain the recurrence and metastasis of tumors. Notably, chemotherapy is often accompanied by a series of adverse reactions, such as short-term nausea, vomiting, etc., and long-term use of taxane drugs may induce neuropathy, or severe side effects such as cardiomyopathy and heart failure caused by HER2-targeted drugs. Statistical data shows that although chemotherapy drugs developed in the past more than 60 years have, to a certain extent, extended the survival period of tumor metastasis patients (about 5 months on average), they have ultimately failed to change their fate of death due to tumor metastasis.

[0004] The research by Morgan et al. (analysis based on 22 clinical trials between 1990 and 2004) revealed that anti-cancer drugs can only increase the 5-year survival rate of tumor patients by 2.1%. Further clinical research by Sharouni et al. found that chemotherapy drugs may accelerate the regeneration of non-small cell lung cancer, and the tumor volume doubling time of patients in the chemotherapy group was significantly shorter than that in the non-chemotherapy group. The research published by Rustin et al. in The Lancet magazine in 2010 pointed out that there was no significant difference in the median survival period between early chemotherapy and delayed chemotherapy for ovarian cancer patients, and the median survival period of the early chemotherapy group was even shortened by 2 months, which strongly suggests that the toxic effects of chemotherapy drugs cannot be ignored. Generally speaking, the unsatisfactory treatment effects of current anti-cancer drugs can be attributed to the serious toxicity of the drugs themselves, the decline in the quality of life of patients, the damage to the immune system and recovery ability due to anti-cancer treatment, and the limitations of traditional cancer prevention and treatment strategies, etc. Therefore, there is an urgent need to develop new technologies and products that can effectively prevent tumor recurrence and metastasis to alleviate human fear of tumor metastasis and reduce related mortality. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of glehnol in the preparation of drugs.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides an application of phellopterin in the preparation of drugs, and an application of phellopterin in the preparation of drugs for preventing recurrence and metastasis after tumor surgery; the chemical structural formula of the phellopterin is shown in the following formula 1:

[0008]

[0009] Further, the concentration of the phellopterin is 1-100 μM.

[0010] Further, the application of the phellopterin as a single dosage form or in combination with other drugs in the preparation of drugs for preventing recurrence and metastasis after tumor surgery.

[0011] Further, the application of a composition formed by combining the phellopterin with an anti-cancer drug in the preparation of drugs for preventing recurrence and metastasis after tumor surgery.

[0012] Further, the drug is an oral dosage form, including one or more of tablets, lozenges, troches, aqueous or oily suspensions, powders or granules, emulsions, hard capsules or soft capsules, solutions, syrups and elixirs.

[0013] Compared with the prior art, the present invention discovers a new application of phellopterin. By applying phellopterin in the preparation of drugs for preventing recurrence and metastasis after tumor surgery, at a concentration that does not cause cytotoxicity and side effects, through intervening in multiple key steps of tumor metastasis, including inhibiting the adhesion of circulating tumor cells to vascular endothelial cells, inhibiting the EMT transformation of circulating tumor cells, inhibiting the transendothelial migration of circulating tumor cells, etc., the effect of preventing tumor metastasis or treating tumors is achieved. At the same time, the activity of immune cells is enhanced, the killing ability of immune cells against circulating tumor cells is increased, and the survival period is prolonged. It has extremely high safety and solves the problems such as the impairment of the immune system and the rehabilitation ability caused by traditional drugs in anti-cancer treatment and the limitations of traditional cancer prevention and treatment strategies.

[0014] Description of the drawings

[0015] Figure 1 It is a schematic diagram of the results of the effect of phellopterin on the adhesion of cancer cells to human umbilical vein endothelial cells HUVECs in Example 2;

[0016] Figure 2 It is a schematic diagram of the results of the effect of phellopterin on the expression of adhesion proteins ICAM-1, VCAM-1 and E-selectin on the surface of HUVECs induced by the inflammatory factor TNF-α in Example 3;

[0017] Figure 3 It is a schematic diagram of the results of the inhibition of tumor cell metastasis by phellopterin in Example 4;

[0018] Figure 4 Schematic diagram of the results of the inhibition of EMT transformation-related protein by Glehnol in Example 5.

[0019] Figure 5 Schematic diagram of the results of the inhibition of lung metastasis nodules of mouse breast cancer by Glehnol in Example 6.

[0020] Figure 6 Schematic diagram of the H&E staining results of the lung, liver, spleen, kidney and heart sections of mice after treatment with Glehnol in Example 6.

[0021] Figure 7 Schematic diagram of the results of the significant increase in anti-tumor immune cells, cytotoxic T cells (Tc cells), by Glehnol in Example 7.

[0022] Figure 8 Schematic diagram of the results of the significant increase in anti-tumor immune cells, natural killer cells (NK cells), by Glehnol in Example 7. Detailed implementation manners

[0023] The technical solutions of the present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only for exemplarily illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0024] Example 1

[0025] Use of Glehnol at a concentration of 1-100 μM in the preparation of a drug for preventing recurrence and metastasis after tumor surgery; the chemical structural formula of Glehnol is shown as Formula 1 below:

[0026]

[0027] Among them, when Glehnol is used as a single dosage form or in combination with other drugs in the preparation of a drug for preventing recurrence and metastasis after tumor surgery to comprehensively and comprehensively control all links of tumor metastasis, the drug is an oral dosage form, including one or more of tablets, lozenges, troches, water or oil suspensions, powders or granules, emulsions, hard capsules or soft capsules, solutions, syrups and elixirs.

[0028] When Glehnol is used as a single drug, the dosage level is about 0.001 mg / kg to 50 mg / kg body weight per day. To achieve the purpose of preventing the recurrence of primary tumors in mammalian patients after surgical resection and metastasis, the daily dose is usually about 0.005 mg / kg to 5 mg / kg.

[0029] In one embodiment, for the prevention and treatment of diseases, the medicament of the present invention can be formulated with other pharmaceutically acceptable carriers into dosage forms for orally, topically, parenterally, by inhalation, by spraying, rectally or vaginally administering the active ingredient(s) (alone or in combination). Parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection, intracisternal injection or infusion methods.

[0030] In one embodiment, phellopterin can be prepared into a composition for oral use according to any method known in the field of pharmaceutical composition preparation. Such compositions usually contain one or more excipients and pharmaceutical carriers selected from sweeteners, flavoring agents, coloring agents, preservatives, etc. The excipient can be a diluent such as lactose, calcium carbonate, sodium carbonate, calcium phosphate or sodium phosphate; a granulating agent and disintegrant such as corn starch or alginic acid; a binder such as starch, gelatin or gum arabic; and a lubricant such as magnesium stearate, stearic acid or talc. Tablets can be uncoated or can be coated. Coating can delay the decomposition of the drug in the gastrointestinal tract, thereby prolonging the action time of the drug. For example, time-delay materials such as glyceryl monostearate or glyceryl distearate can be used.

[0031] Example 2

[0032] Phellopterin has the effect of inhibiting the adhesion of tumor cells to vascular endothelial cells.

[0033] Take human umbilical vein endothelial cells (HUVECs) in the logarithmic growth phase, digest them with trypsin, evenly spread them in a 24-well plate, and place them in an incubator at 37 °C and 5% CO 2 for overnight culture. When the cell confluence is over 95%, give TNF-α stimulator (10 ng / mL), and leave a group without TNF-α as a negative control. After 4 h, digest breast cancer cells (MCF-7 cells), stain them with rhodamine and count. Add MCF-7 cells onto HUVECs. After 1 h, wash away the non-adherent cells, and take pictures and count. The experimental method is as follows: 1) Prepare phellopterin solutions with different concentration gradients using ECM medium, which are 0, 1, 10, 50, 100 μM respectively; 2) Digest MCF-7 cells, collect cell precipitates, and wash them 2 times with PBS; 3) Resuspend the cells with 1 mL PBS and add 1 μL rhodamine and mix well, incubate in the dark at room temperature for 15 min; 4) Centrifuge the cells, wash them 2 times with PBS, and collect cell precipitates and resuspend them with ECM; 5) Adjust the cell concentration to 5×105 cells / mL with the above different concentration phellopterin solutions; 6) Add 100 μL of the above cell suspension into HUVECs cells stimulated with TNF-α for 4 h, and continue to culture in an incubator at 37 °C and 5% CO 2 saturated humidity for 1 h; 7) Remove the non-adherent MCF-7 cells, wash them 3 times with PBS, take pictures and count using a fluorescence microscope, and calculate the adhesion inhibition rate of phellopterin on tumor cells.

[0034] The results are as Figure 1 shown. The adhesion rate between tumor cells and endothelial cells shows a dose-effect relationship with the concentration of Glehnol. Compared with the blank control group, as the drug concentration increases, the adhesion ability of tumor cells on the surface of endothelial cells decreases.

[0035] Example 3

[0036] Glehnol inhibits the expression of adhesion proteins ICAM-1, VCAM-1, and E-selectin on the surface of vascular endothelial cells.

[0037] The relationship between Glehnol and the expression of adhesion molecules (CAMs) on the surface of HUVECs was detected by flow cytometry to detect the expression levels of VCAM-1 (CD106), ICAM-1 (CD54), and E-selectin (CD62E). The experimental method is as follows: 1) Prepare Glehnol solutions with different concentration gradients using ECM medium, which are 0, 0, 1, 10, 50, and 100 μM respectively; 2) Seed HUVECs endothelial cells in 6-well plates. When the cell confluence is above 60%, add the above different concentration Glehnol solutions and continue culturing in an incubator; 3) After 20 h, stimulate with TNF-α (10 ng / mL), and the negative control wells are not stimulated; 4) After continuing to culture for 4 h, digest the cells with trypsin without EDTA, wash twice with PBS, and collect the cell precipitate; 5) Resuspend the cells in 200 μL PBS, stain with CD54-APC, CD106-PE, and CD62E-APC antibodies respectively, and incubate at 4 °C in the dark for 30 min; 6) Centrifuge to collect the cell precipitate, wash twice with PBS, resuspend the cells with 500 μL, perform flow cytometry detection, and use FlowJo 10.6.2 software for data analysis of the expression of CD54, CD106, and CD62E.

[0038] The results are as Figure 2 shown. Glehnol has a significant inhibitory effect on the expression of adhesion molecules on the surface of TNF-α-induced HUVECs, achieving the effect of inhibiting circulating tumor cells from adhering to the vascular intima. Compared with the control group, as the concentration of Glehnol increases, Glehnol has a dose-dependent inhibitory effect on the expression of VCAM-1, ICAM-1, and E-selectin on the surface of TNF-α-induced endothelial cells.

[0039] Example 4

[0040] Glehnol inhibits tumor cell metastasis by regulating the THBS1 and KITLG signaling pathways of tumor cells.

[0041] MCF-7 cells were seeded in 6-well plates. When the cell confluence reached 40 - 60%, phellopterin was administered at concentrations of 0, 10, 50, and 100 μM. After administration, the plates were placed in an incubator at 37°C and 5% CO 2 saturated humidity and cultured for another 24 h, then total proteins were extracted for Western Blot assay.

[0042] The results were as Figure 3 shown that phellopterin could reduce the expression of tumor cell THBS1, KITLG, and TRIM22, inhibit the THBS1 and KITLG signaling pathways of tumor cells, reduce the motility and migration ability of tumor cells, and thus significantly inhibit the transendothelial migration ability of tumor cells.

[0043] Example 5

[0044] Phellopterin inhibits EMT transformation of tumor cells.

[0045] MCF-7 cells were seeded in 6-well plates. When the cell confluence reached 40 - 60%, phellopterin was administered at concentrations of 0, 10, 50, and 100 μM. After administration, the plates were placed in an incubator at 37°C and 5% CO 2 saturated humidity and cultured for another 24 h, then total RNA was extracted for qRT-PCR assay. The specific method was as follows: 1) Remove the culture medium, wash twice with PBS, add 1 mL of Trizol solution, and pipette vigorously until the cells were completely lysed, then collect in a centrifuge tube; 2) Add 200 μL of chloroform, shake vigorously for 30 s, and let stand at room temperature for 5 min; 3) Centrifuge at 12000 rpm at 4°C for 10 min; 4) Gently aspirate the supernatant, add an equal volume of isopropanol, mix gently, and let stand for 10 min; 5) Centrifuge at 12000 rpm at 4°C for 15 min to remove the supernatant; 6) Add 1 mL of pre-cooled 75% ethanol to suspend the RNA precipitate, centrifuge at 7500 rpm at 4°C for 5 min, and dissolve with an appropriate amount of DEPC water; Measure the RNA concentration and purity with a micro ultraviolet spectrophotometer; 7) Reverse transcribe the RNA into cDNA according to the instructions, and the specific dosage refers to Table 1; 8) Perform real-time quantitative PCR reaction according to the kit instructions and analyze the relative expression of mRNA.

[0046] The results were as Figure 4 shown that compared with the control group, phellopterin could reduce the mRNA and protein levels of EMT transcription factors SNAIL, TWIST, and ZEB, and reduce the mRNA levels of mesenchymal phenotype FN1 and CDH2 and the protein levels of N-cadherin and β-catenin, thereby inhibiting EMT transformation of tumor cells and reducing the migration and invasion ability of tumor cells.

[0047] Example 6

[0048] Glehnol inhibits the pulmonary metastasis of breast cancer.

[0049] After the mouse breast cancer cells 4T1 were amplified and cultured in vitro, they were digested into single cells with trypsin and resuspended in PBS at a density of 5×10 5 cells / mL. 100 μL of the cell suspension was injected into the tail vein to establish a mouse breast cancer metastasis model. The mice were intraperitoneally injected with glehnol at doses of 1 mg / kg, 5 mg / kg, and 10 mg / kg daily. After 21 days, the mice were sacrificed, and the lung tissues were removed, fixed with Brin's stain overnight, the number of lung nodules was recorded, and the pathological changes were observed by HE staining.

[0050] The results were as Figure 5 shown. The number of pulmonary tumor metastasis nodules in the mice treated with glehnol was significantly less than that in the control group, and the number of nodules showed a dose-dependent relationship with glehnol. Compared with the normal group, the weight of the lung tissue in the tumor group increased, but it tended to return to normal after treatment with glehnol.

[0051] The results of HE staining are shown in Figure 6 . Compared with the tumor group, the tumor nodules in the lung tissue were significantly reduced or disappeared after treatment with glehnol, especially in the higher-dose group, which had basically returned to normal. In addition, through the HE staining results of the heart, liver, spleen, lungs, and kidneys, it was found that glehnol did not produce obvious toxic and side effects, further demonstrating the safety of glehnol.

[0052] Example 7

[0053] Effect of glehnol on immune cells in the blood of mice.

[0054] Referring to the method in Example 6, before sacrificing the mice, blood was collected from the eye sockets into heparin-containing centrifuge tubes. Erythrocyte lysate was added, and after lysing at 37 °C for 10 min, centrifugation was immediately carried out at 1000 rpm for 5 min. The cell pellet was collected, washed twice with pre-cooled PBS, CD45-V450, CD3-PerCP-Cy5.5, and CD8-PE-Cy7 antibodies were added, and incubation was carried out at 4 °C in the dark for 30 min. Centrifugation was carried out at 1000 rpm for 5 min, the cell pellet was collected, washed twice with pre-cooled PBS, resuspended in PBS, and detected by flow cytometry.

[0055] The results were as Figure 7 and Figure 8 shown. The proportions of Tc cells (i.e., CD3+CD8+ positive cells) and NK cells in the blood of the tumor group mice were significantly lower than those in the normal group, while in the glehnol treatment group, the proportions of Tc and NK cells gradually increased and approached the levels of normal healthy mice, indicating that glehnol has the effect of enhancing the activity of immune cells in mice, enhancing the ability of immune cells to kill circulating tumor cells, and achieving the effect of preventing tumor metastasis.

[0056] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments we described are illustrative rather than used to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the scope of the patent application of the present invention should be covered by the scope protected by the claims of the present invention.

Claims

1. The use of anthocyanin in the preparation of medicines, characterized in that: The application of coral acanthion in the preparation of a drug for preventing postoperative tumor recurrence and metastasis; the chemical structure of the coral acanthion is shown in Formula 1 below:

2. The use of anthocyanin in the preparation of medicines according to claim 1, characterized in that: The concentration of the anthocyanin is 1-100 μM.

3. The use of anthocyanin in the preparation of medicines according to claim 1, characterized in that: The anthocyanin is used as a single dosage form or in combination with other drugs in the preparation of drugs for preventing postoperative tumor recurrence and metastasis.

4. The use of anthocyanin in the preparation of medicines according to claim 1, characterized in that: The composition composed of the coral acanthocyanin and anticancer drugs is used in the preparation of drugs for preventing postoperative recurrence and metastasis of tumors.

5. The use of anthocyanin in the preparation of medicines according to claim 1, characterized in that: The drug is in oral dosage form, including one or more of tablets, lozenges, troches, aqueous or oily suspensions, powders or granules, emulsions, hard or soft capsules, solutions, syrups and elixirs.

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