阅读说明：本技术 一种含羟基的化合物在制备药物中的应用 (Application of hydroxyl-containing compound in preparation of medicine ) 是由 吴若铭 叶冠 王辉俊 吕兴 于 2019-09-18 设计创作，主要内容包括：本发明公开了一种含羟基的化合物在制备药物中的应用。本发明提供了一种如式I所示含羟基的化合物在制备抗神经胶质瘤药物中的应用。本发明还提供了一种如式I所示含羟基的化合物在制备细胞周期蛋白D1抑制剂和细胞周期蛋白E抑制剂的应用以及制备Jak蛋白磷酸化抑制剂和Stat3蛋白磷酸化抑制剂的应用。本发明的含羟基的化合物能有效抑制神经胶质瘤细胞U251的增殖,为研究与开发新的抗神经胶质瘤药物提供了候选药物,为开发利用桃金娘中的天然活性物质提供了科学依据。(The invention discloses an application of a compound containing hydroxyl in preparing a medicament. The invention provides application of a hydroxyl-containing compound shown as a formula I in preparation of an anti-glioma drug. The invention also provides application of the hydroxyl-containing compound shown as the formula I in preparation of cyclin D1 inhibitors and cyclin E inhibitors and application in preparation of Jak protein phosphorylation inhibitors and Stat3 protein phosphorylation inhibitors. The hydroxyl-containing compound can effectively inhibit the proliferation of glioma cells U251, provides a candidate drug for researching and developing a new glioma-resisting drug, and provides a scientific basis for developing and utilizing natural active substances in myrtle.)

1. An application of a hydroxyl-containing compound shown as a formula I or a pharmaceutically acceptable salt thereof in preparing a drug for resisting glioma;

2. the use of claim 1, wherein the glioma is diffuse astrocytoma, oligodendroglioma or ependymoma.

3. A pharmaceutical composition comprises a compound containing hydroxyl as shown in formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutic adjuvant;

4. the pharmaceutical composition of claim 3, wherein the pharmaceutical composition is an anti-glioma pharmaceutical composition.

5. The pharmaceutical composition of claim 3 or 4, wherein the hydroxy-containing compound of formula I or a pharmaceutically acceptable salt thereof is used in a therapeutically effective amount.

6. The pharmaceutical composition of claim 3 or 4, wherein the pharmaceutical composition is in the form of an oral, vaginal, anal, nasal, intramuscular or intravenous formulation.

7. The pharmaceutical composition of claim 6, wherein: the oral preparation is tablet, capsule, granule, sustained release preparation or dripping pill.

8. An application of a hydroxyl-containing compound shown as a formula I or a pharmaceutically acceptable salt thereof in preparing a U251 cell proliferation inhibitor;

9. an application of a compound containing hydroxyl as shown in formula I or pharmaceutically acceptable salt thereof in preparing cyclin D1 inhibitor and cyclin E inhibitor;

10. an application of a hydroxyl-containing compound shown as a formula I or a pharmaceutically acceptable salt thereof in preparing a Jak protein phosphorylation inhibitor and a Stat3 protein phosphorylation inhibitor;

Technical Field

The invention relates to application of a compound containing hydroxyl in preparing a medicament.

Background

Gliomas are the most common of the tumors of the central nervous system. The pathogenesis of glioma is still unclear. Its occurrence and development may be related to physical factors, external environmental factors, and gene mutations. It is the result of the co-participation of multiple protooncogenes and tumor suppressor genes. In recent years, with the rapid development of modern biomedicine, research on the pathogenesis and etiology of glioma has been advanced to some extent. Studies have shown that glioma development may be associated with chromosomal mutations, deletions and mutations in tumor suppressor genes, and rearrangement and amplification of oncogenes. There are two genes that are closely related to the pathogenesis of tumors: one is a protooncogene and the other is an anti-tumor gene. Activation, over-expression and limited expression of proto-oncogenes may induce tumorigenesis. In contrast, the presence and normal expression of anti-tumor genes contribute to the suppression of tumorigenesis. The protooncogene is present in normal cells of the human body, and under normal conditions, the protooncogene is not expressed or expressed, and does not induce canceration of the cells. Under the stimulation of chemical products, viral biologicals, radioactive substances, etc., protooncogenes in normal cells in the body may be abnormally activated. The expression of protooncogenes evades immune surveillance by the body and the number of tumor cells rapidly increases, leading to the formation of tumor tissue. Despite the widespread clinical use of surgical resection, radiation therapy and chemotherapy, the average annual survival rate of glioblastoma patients is only 46%. One of the reasons for this is that the mechanism of development of glioma is not yet known. Therefore, the basic research on the pathogenesis of glioma has important scientific and social significance for treating the disease and improving the survival rate. Human glioma cell U251, growing invasively, is multinucleated, and its carcinogenic glioma is considered to be one of the most malignant tumors in human tumor cells. Research shows that the proliferation of glioma cells can be inhibited through inhibiting Jak/Stat3 signal channels and cell cycles, the apoptosis process is influenced, and the growth of tumors is inhibited.

Cyclin D1 is an important cyclin that plays an important role in cell cycle progression. Recent studies have found that cyclin D1 is involved in the regulation of prostate and oral squamous cell carcinoma cell proliferation. Cyclin E is a G1-phase nucleoprotein originally extracted from Saccharomyces cerevisiae. In normal cells, cyclin E begins to be synthesized in G1 and reaches its highest level in S phase. Degradation occurs in S phase, and G2 phase and M phase are not expressed. In abnormal cases, the disordered over-expression of cyclin E throughout the cell cycle leads to abnormal proliferation of cells beyond control, resulting in tumorigenesis.

Myrtle (Rhodomyrtus tomentosa) is an evergreen shrub originated from southeast asia, and is used in traditional thailand medicine for treating large intestine diarrhea, dysentery, abscess, hemorrhage and gynecological diseases. In addition, it is also used for preparing whitening, anti-aging and skin cosmetic agents. The traditional medical activity of this plant may be due to its antioxidant activity. Several compounds have been isolated so far from the leaves of this plant, such as Rhodomyrtone, anthocyanidins, flavanic acid derivatives and hexacyclic chlorothioglucitol derivatives (tominosonea and B). Despite extensive phytochemical research, there is no systematic way to understand the immunopharmacological activity of such plants or their components.

At room temperature through CH₂Cl₂，Me₂CO and MeOH were used to extract the dried leaves of the myrtle continuously. Mixing Me with water₂The CO extract was dissolved in hexane and fractionated to obtain soluble and insoluble fractions. Then, the soluble fraction was separated by QCC and hexane-CH was used₂Cl₂、CH₂Cl₂、CH₂Cl₂-Me₂CO and Me₂CO gradient solvent was eluted multiple times to obtain Rhodomyrtone.

Disclosure of Invention

The invention aims to overcome the defect of single type of the existing drugs for treating glioma, and provides application of a hydroxyl-containing compound in preparing drugs. The hydroxyl-containing compound Rhodomyrtone can effectively inhibit the expression of cyclin D1 and cyclin E and the phosphorylation of Jak and Stat3 proteins, and can be used for preparing anti-glioma drugs.

The invention solves the technical problems through the following technical scheme.

The invention provides an application of a hydroxyl-containing compound shown as a formula I or pharmaceutically acceptable salt thereof in preparing a medicament for resisting glioma;

the glioma is preferably diffuse astrocytoma, oligodendroglioma and ependymoma.

The invention also provides a pharmaceutical composition, which comprises the hydroxyl-containing compound shown as the formula I or pharmaceutically acceptable salt thereof and pharmaceutic adjuvant;

the invention provides an anti-glioma pharmaceutical composition, which comprises a hydroxyl-containing compound shown as a formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutical adjuvant;

the invention provides a pharmaceutical composition, wherein the dosage of the hydroxyl-containing compound shown as the formula I or the pharmaceutically acceptable salt thereof is therapeutically effective amount;

the dosage form of the pharmaceutical composition can be oral, vaginal, anal, nasal, muscular or intravenous preparations.

The oral preparation is tablet, capsule, granule, sustained release preparation or dripping pill.

The invention provides an application of a hydroxyl-containing compound shown as a formula I or a pharmaceutically acceptable salt thereof in preparing a U251 cell proliferation inhibitor;

the invention provides application of a hydroxyl-containing compound shown as a formula I or pharmaceutically acceptable salt thereof in preparation of a cyclin D1 inhibitor and a cyclin E inhibitor;

the invention provides application of a hydroxyl-containing compound shown as a formula I or pharmaceutically acceptable salt thereof in preparation of a Jak protein phosphorylation inhibitor and a Stat3 protein phosphorylation inhibitor;

the reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the Rhodomyrtone can effectively inhibit the expression of cyclin D1 and cyclin E and the phosphorylation of Jak and Stat3 proteins, provides a candidate drug for researching and developing a new anti-glioma drug, and provides a scientific basis for developing and utilizing natural active substances in myrtle.

Drawings

FIG. 1 shows that Rhodomyrtone inhibits proliferation of U251 cells. Cell viability after 24 hours treatment with 1, 3, 9 μ M Rhodomyrtone, respectively, indicates P < 0.05 compared to the blank control and DMSO groups.

FIG. 2 shows that Rhodomyrtone inhibits proliferation of U251 cells. Cell viability after 48 hours of treatment with 1, 3, 9 μ M Rhodomyrtone, respectively, indicated P < 0.05 compared to the blank control and DMSO groups.

FIG. 3 shows the cell distribution of the control group at different cell cycle stages.

FIG. 4 shows Rhodomyrtone blocking U251 cells at G0/G1. Cell distribution of U251 cells of different cell cycles treated with 3. mu.M Rhodomyrtone for 24 hours.

FIG. 5 shows Rhodomyrtone blocking U251 cells at G0/G1. Cell distribution of U251 cells in different cell cycles at 48h treated with 3. mu.M Rhodomyrtone.

FIG. 6 shows Rhodomyrtone blocking U251 cells at G0/G1. The cell fraction of U251 cells at different cell cycle stages of each group, 24h and 48h, respectively, was treated with 3. mu.M Rhodomyrtone, compared to the DMSO group.

FIG. 7 is a graph showing that Rhodomyrtone inhibits the Jak/Stat signaling pathway in U251 cells. The expression levels of p-Jak3, p-Stat3, Stat3 and internal reference protein Tubulin at 15min, 30min, 1h and 2h, respectively, were treated with 3. mu.M Rhodomyrtone, compared to the DMSO group.

FIG. 8 shows that Rhodomyrtone inhibits cyclin D1 and cyclin E expression in U251 cells. The expression levels of Cyclin D1, Cyclin E and internal control protein Tubulin were compared with those in the DMSO group at 15min, 30min, 1h and 2h, respectively, with 3. mu.M Rhodomyrtone.

Detailed Description

The present invention will be explained in detail with reference to examples, but the scope of the present invention is not limited thereto. The experimental procedures not described in detail in the experiments are all routine experimental procedures well known to those skilled in the art.

Example 1: preparation of compound Rhodomyrtone

Heating and refluxing 10Kg of dried branches and leaves of myrtle with 90% ethanol (the volume ratio of the dried branches and leaves to the medicinal materials is 8: 1) for 3 times, 1 hour each time, concentrating under reduced pressure to obtain an extract, extracting with petroleum ether and ethyl acetate to obtain an ethyl acetate part, mixing the ethyl acetate part with 100-200 meshes of silica gel according to the mass ratio of 1: 1, carrying out column chromatography on 200-300 meshes of silica gel, taking a Rhodomyrtone sample as a reference substance, carrying out TLC (thin layer chromatography) on eluent to obtain Rhodomyrtone part eluent, merging and concentrating, adding an appropriate amount of acetone for recrystallization to obtain a Rhodomyrtone crude product681 mg. About 150mg of crude Rhodomyrtone was purified by Shimadzu LC-20AD HPLC, Zorbax SB-C18 silica gel column (9.4X 250mm, 5 μ M), SPD-M20A UV detector, mobile phase in methanol: semi-preparative was eluted with water (80: 20) to give Rhodomyrtone 100 mg. Rhodomyrtone has a molecular weight of 442 as determined by LC-MS. Rhodomyrtone and CDCl₃And (4) carrying out NMR (nuclear magnetic resonance) on the solution to obtain a C spectrum and an H spectrum, and the C spectrum and the H spectrum are consistent with data reported in the literature. The nmr data are shown in table 1.

TABLE 1 NMR data for the compound Rhodomyrtone

Structural formula of Rhodomyrtone

Example 2: anti-glioma biological activity of compound Rhodomyrtone and related mechanism

1. Experimental materials and methods

Glioma cell line U251 was purchased from Shanghai institute of cell biology, Chinese academy of sciences. Glioma cells were cultured in medium containing fetal bovine serum (10%) and streptomycin (100. mu.g/ml) penicillin (100 IU/ml). The flasks were placed at 37 ℃ and 5% CO₂In an incubator. The medium was changed every 24-48 hours depending on the growth of the cells, the color and turbidity of the medium. When the cells grew to more than 70% of the bottom of the flask, passaging was performed to adjust the cell density in the flask.

Cell proliferation rate was measured by cell counting kit 8(CCK 8). The cell suspension was seeded into 96-well plates (100. mu.L/well). Plates were incubated at 37 ℃ with 5% CO₂Pre-culturing in the incubator. To each well was added a CCK8 solution. The incubation plate is incubated in an incubator for 1-4 hours. Enzyme labelThe method measures the absorption at 450 nm.

Cell survival (%) ([ a (drug) -a (blank) ]/[ a (0 drug) -a (blank) ] × 100

A (drug): cell, CCK8 solution and drug solution absorption pores

A (blank): cell-free wells were imbibed with medium and CCK8 solution.

A (drug 0): cell uptake wells, CCK8 solution, no drug solution.

Flow cytometry: PI (propidium iodide) penetrates DNA to stain, and cell cycle distribution is detected by flow cytometry. U251 cells were taken 24 hours after drug treatment and washed twice with PBS (phosphate buffered saline). Add pre-cooled 70% methanol, fix at 4 ℃ for more than 4 hours, wash cells twice with PBS, and centrifuge. mu.L of RNaseA (ribonuclease A) was added to each sample, and 400. mu.L of PI was added without light after 30 minutes of immersion at 37 ℃. Flow cytometry examined the cell cycle.

Extracting cell protein: after the incubation was completed, PBS was washed several times. RIPA lysate containing the protease inhibitor PMSF was added and the cells were scraped. The 1 ml syringe was repeatedly aspirated. Cells were placed on ice, spun repeatedly, centrifuged, and then transferred to a new EP tube. Proteins were quantitated using BCA (or directly frozen in-80 ℃ freezer). The concentration was calculated, the protein was dispensed according to the sample size, and soaked in boiling water for 5 minutes. Samples were loaded using high speed transients or frozen directly at-20 ℃ (protein samples frozen at-20 ℃ required re-boiling for 2 minutes before sampling).

Rhodomyrtone-treated cellular proteins and control cellular proteins were collected in RIPA lysate, adjusted to the same loading of 200. mu.g, and after separation of the proteins on a 10% SDS-PAGE gel, the proteins were transferred to a Nitrocellulose (NC) membrane using a BiO-RAD semidry cell (constant pressure 100V, 60 minutes), and the NC membrane was removed. And (5) transferring. At the end of the membrane transfer, the NC membrane was removed and incubated with 5% skim milk powder for 1 hour. The antibody was then added at a 1: 1 dilution and incubated overnight at 4 ℃. The secondary antibody was then added and incubated at 37 ℃ for 1 hour, washed 3 times with 1 × TBST for 5 minutes. ECL luminescence solution was added for 1 minute incubation, exposure, development and fixation.

2. Materials and reagents

3. Results of the experiment

The results of the CCK8 experiment show that the proliferation rate of U251 cells is obviously lower than that of the control group when the Rhodomyrtone is treated for 24 hours and 48 hours. The higher the concentration, the lower the proliferation rate. This indicates that 1, 3, 9 μ M Rhodomyrtone is dose-and time-dependent on proliferation of U251 cells. As shown in fig. 1-2.

For further study of inhibitory effect of Rhodomyrtone on proliferation of U251 cells, the ratio of G0/G1 cells in U251 cells after 24h and 48h of Rhodomyrtone treatment was significantly higher than that in the control group. The number of U251 cells in the G2/M phase was significantly lower than that in the control group. The proportion of U251 cells in the 48h group G0/G1 was higher than in the 24h group, indicating that U251 is mainly arrested in the G0/G1 phase and the number of U251 cell blocks increased with increasing duration of Rhodomyrtone treatment. As shown in fig. 3-6.

In order to clarify a specific mechanism that Rhodomyrtone influences the U251 cell cycle and proliferation, Western blotting is adopted to detect the expression of related proteins after Rhodomyrtone treatment. The Jak/Stat3 signaling pathway as well as Cyclin D1 and Cyclin E were examined over time in 3. mu.M Rhodomyrtone-treated U251 cells. The results show that with increasing Rhodomyrtone treatment time, the expression of cyclinD1 and cyclinE decreased, the expression of p-Jak (phosphorylated Jak protein) and p-Stat3 (phosphorylated Stat3 protein) decreased, and the inhibition of Jak/Stat3 signaling pathway increased. As shown in fig. 7-8.

The above experiments clearly show that Rhodomyrtone can effectively inhibit proliferation of U251 cells and block the G0/G1 phase in a dose-dependent and time-dependent manner. Meanwhile, Rhodomyrtone can also inhibit Jak/Stat3 signals and inhibit the expression of Cyclin D1 and Cyclin E proteins. Therefore, Rhodomyrtone can be used for preparing anti-glioma drugs. The invention provides a candidate drug for researching and developing a new anti-glioma drug and provides a scientific basis for developing and utilizing natural active substances in the myrtle.