Pharmaceutical use of three compounds in selaginella martensii

Abstract

The present application provides medical uses of various compounds in Selaginella martensii, specifically, the use in preparing anti-melanoma or breast cancer drugs.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry. Background Technology

[0002] Selaginella tamariscina (P. Beauv.) Spring is a terrestrial or lithophytic fern belonging to the genus Selaginella in the family Selaginellaceae. It has many forked, densely hairy roots that, together with the stems and branches, form a tree-like trunk; the twigs are sparse and regular, hairless, and flattened dorsoventrally; the leaves are alternately arranged, thick, smooth, with white edges, green or brown, and finely toothed; the megaspores are pale yellow, and the microspores are orange-yellow; it flowers from July to September; and fruits from September to October. Selaginella gets its name from its stems and leaves, which resemble the young branches and leaves of a cypress tree, and the inward curling of the leaves.

[0003] Selaginella is native to China and prefers warm, humid, and semi-shaded environments. It is mostly found on sunny mountain slopes or in dry rock crevices. Selaginella reproduces by spores.

[0004] According to the "Compendium of Materia Medica," Selaginella tamariscina possesses the effects of promoting blood circulation, regulating menstruation, removing blood stasis, and stopping bleeding. It can be used to treat amenorrhea, dysmenorrhea, abdominal masses, hematemesis, metrorrhagia, hematochezia, rectal prolapse, and other diseases. With its graceful, verdant foliage, Selaginella tamariscina is a beautiful miniature indoor bonsai, evergreen year-round, resembling a sturdy pine tree. It is highly valued for its ornamental qualities, used in rockeries and large bonsai cultivation. Selaginella tamariscina boasts an evolutionary history of 400 million years, making it one of the oldest groups of terrestrial plants discovered to date. Summary of the Invention

[0005] This invention provides the use of one or more of the following compounds in the preparation of drugs for treating melanoma or triple-negative breast cancer;

[0006] The compounds are: diselaginellins A, diselaginellins B, and Selaginellin A.

[0007]

[0008] The present invention also provides an antitumor drug composition comprising any one or more of diselaginellins A and diselaginellins B.

[0009] Melanoma, usually referring to malignant melanoma, is a highly malignant tumor that arises from the malignant transformation of melanocytes. Malignant melanoma commonly occurs in the skin, but can also occur in mucous membranes (including visceral mucous membranes), the uvea of ​​the eye, and the pia mater. DNA damage to melanocytes is the fundamental cause of malignant melanoma. Against a genetic background, prolonged exposure to ultraviolet radiation, repeated friction, and external trauma can lead to DNA breaks, translocations, mutations, or abnormal methylation in melanocytes. This results in uncontrolled proliferation and abnormal differentiation of melanocytes, ultimately developing into malignant tumor cells.

[0010] Breast cancer is a condition in which breast epithelial cells proliferate uncontrollably under the influence of various carcinogenic factors. Early symptoms often include breast lumps, nipple discharge, and enlarged axillary lymph nodes. In later stages, distant metastasis of cancer cells can lead to multi-organ complications, directly threatening the patient's life.

[0011] Triple-negative breast cancer refers to breast cancer in which the immunohistochemical examination of the cancer tissue shows negative results for estrogen receptor (ER), progesterone receptor (PR), and the proto-oncogene Her-2. This type of breast cancer accounts for 10.0% to 20.8% of all breast cancer pathological types. It has unique biological behavior and clinicopathological features, and its prognosis is worse than other types, earning it the nickname "King of Breast Cancers." Experiments have shown that the drug of this invention can effectively inhibit the growth of this type of cancer cell.

[0012] The pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0013] The pharmaceutically acceptable excipients described in this invention refer to all additional materials in a drug other than the active pharmaceutical ingredient (API). Excipients should possess the following properties: (1) non-toxic to the human body and with virtually no side effects; (2) chemically stable and not easily affected by temperature, pH, storage time, etc.; (3) without incompatibility with the API and without affecting the efficacy and quality control of the API; and (4) not interacting with packaging materials. In this invention, excipients include, but are not limited to, fillers (diluents), lubricants (flow aids or anti-adhesion agents), dispersants, wetting agents, adhesives, regulators, solubilizers, antioxidants, antibacterial agents, emulsifiers, disintegrants, etc. Binders include syrups, gum arabic, gelatin, sorbitol, astragalus gum, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose, or hydroxypropyl methyl cellulose), gelatin paste, syrup, starch paste, or polyvinylpyrrolidone, etc.; fillers include lactose, powdered sugar, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salts (such as calcium sulfate, calcium phosphate, dicalcium phosphate, precipitated calcium carbonate, etc.), sorbitol, or glycine, etc.; lubricants include micronized silica gel, magnesium stearate, talc, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, etc.; disintegrants include starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate, etc.). The ingredients include: pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.; polyvinylpyrrolidone or microcrystalline cellulose, etc.; humectants include sodium dodecyl sulfate, water or alcohol, etc.; antioxidants include sodium sulfite, sodium bisulfite, sodium metabisulfite, dibutylbenzoic acid, etc.; antibacterial agents include 0.5% phenol, 0.3% cresol, 0.5% chlorobutanol, etc.; regulators include hydrochloric acid, citric acid, potassium hydroxide (sodium), sodium citrate and buffers (including sodium dihydrogen phosphate and disodium hydrogen phosphate), etc.; emulsifiers include polysorbate-80, sorbitan, prorhonic F-68, lecithin, soybean lecithin, etc.; solubilizers include Tween-80, bile, glycerin, etc.

[0014] The tumors described in this invention include primary cancer or metastatic cancer.

[0015] The primary cancer mentioned in this invention refers to the normal cells of originally normal tissues and organs that gradually transform into malignant tumor cells under the long-term effects of various internal and external carcinogenic factors, and then form a mass of cancer cells, namely "primary cancer" or "primary malignant tumor".

[0016] Metastatic cancer, as described in this invention, refers to tumor cells that have invaded lymphatic vessels, blood vessels, or other pathways from the primary site and been carried to other locations to continue growing, forming a tumor of the same type as the primary tumor. This process is called metastasis, and the resulting tumor is called a metastatic tumor or metastatic cancer. Metastasis is a characteristic of malignant tumors.

[0017] Furthermore, the drug is a drug that promotes apoptosis in cancer cells.

[0018] Furthermore, the drug is a drug that induces G2 / M phase arrest in cancer cells.

[0019] The G2 / M phase is one of the two checkpoints in the cell cycle and is an important stage of cell proliferation. Various DNA damaging agents can cause not only G1 phase arrest but also G2 / M phase arrest, and G2 / M phase arrest may be more prevalent.

[0020] Cell cycle checkpoints are crucial control mechanisms in eukaryotic cells to ensure the integrity of chromosome number and the normal functioning of the cell cycle. While normal cells possess multiple cell cycle checkpoints, tumor cells commonly exhibit G1 phase defect, and after DNA damage, they tend to selectively remain in the G1 phase. Therefore, the G1 / M phase checkpoint has become an attractive target for cancer therapy. Prolonged arrest of tumor cells in the G1 / M phase can inhibit tumor growth and induce apoptosis due to accumulated DNA damage, thus achieving a therapeutic effect. However, the prerequisite for this treatment strategy is to employ effective methods to arrest tumor cells in the G1 / M phase. Attached Figure Description

[0021] Figure 1 Cell proliferation inhibition rate (40 μM) of 24 monomeric compounds after treatment with HepG2 hepatocellular carcinoma. Note: Compared with the blank control group, **P<0.01

[0022] Figure 2 Cell proliferation inhibition rate (40 μM) of 24 monomeric compounds after treatment with melanoma A375 cells. Note: Compared with the control group, **P<0.01

[0023] Figure 3 Cell proliferation inhibition rate (40 μM) of 24 monomeric compounds after treatment with mouse melanoma B16-F10 cells. Note: Compared with the control group, *P<0.05, **P<0.01

[0024] Figure 4 The effect of diselaginellins B on the apoptosis rate of MDA-MB-231 cells was detected by flow cytometry (36 h).

[0025] Note: Compared with the blank control group, **P<0.01

[0026] Figure 5 Flow cytometry analysis of the effect of diselaginellins B on the cell cycle of MDA-MB-231 cells

[0027] Figure 6Western blot analysis of the effect of diselaginellins B on the expression of G2 / M cycle-related proteins in MDA-MB-231 cells.

[0028] Figure 7 Western blot analysis of the effect of diselaginellins B on the relative expression of G2 / M cyclins in MDA-MB-231 cells. (n=3), compared with the blank control group * P < 0.05, ** P < 0.01, *** P < 0.001

[0029] Figure 8 Effects of diselaginellins B on intracellular reactive oxygen species (ROS) in MDA-MB-231 cells. Note: **P<0.01 compared with the blank control group. Detailed Implementation

[0030] Example 1: Screening study of the in vitro antitumor activity of the traditional Chinese medicine Selaginella tamariscina.

[0031] 1.1 Experimental Materials

[0032] Tested monomeric compounds: uncinataflavone (1), uncinataflavone A(2), 3,5,7-trimethoxyflavone-4′-O-β-D-glucopyranoside(3), 3,7-dimethoxy-5,4′-dihydroxyflavone(4), 3,5,7-trimethoxyflavone-4′-hydroxyflavone(5), 3,5,7-trimethoxyflavone-4′-O-[5″’-Op-coumaroyl -β-D-apiofuranoyl-(1”’→2”)-β-D-glucopyranoside](6), kaempferol-3-O-α-L-rhamnopyranoside(7), apigenin-7-O-β-D-glucoside(8), kaempferol3-O-(6”-O-acetyl)-β-D-glucopyranoside(9), tamariscin A(10), selaginellin A(11), selaginellin E(12), selaginellin J(13), selaginellin H(14), (R)-selaginellin U(15), (R)-selaginellin V(16), selaginpulvilin A(17), diselaginellins A(18), diselaginellins B(19), selariscinin I(20), selaginellin T(21), selaginellin M(22), selaginellin(23), and selaginellin U(24) were all prepared in the laboratory with a purity of over 98%.

[0033] Table 1. Sources of tumor cell lines

[0034]

[0035] 1.2 Experimental Methods

[0036] 1.2.1 MTT assay for the inhibitory activity of Selaginella monomeric compounds against three types of tumor cells

[0037] Human hepatocellular carcinoma HepG2 cells, human melanoma A375 cells, and mouse melanoma B16-F10 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum for 2-3 days in a 5% CO2 incubator at 37°C.

[0038] Four types of tumor cells in the logarithmic growth phase were collected, the culture medium was discarded, and the cells were washed twice with 2 mL of PBS buffer. 1 mL of trypsin was added, and the cells were gently pipetted. 2 mL of culture medium was added to terminate the digestion. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded. An appropriate amount of fresh culture medium was added, and cell counting was performed. The cells were mixed, and 100 μL of cell suspension was added to each well. Cells were seeded in 96-well plates (1×10⁶ cells / wells). 4 The samples were incubated in an incubator for 12 hours. The supernatant was removed, and each drug was divided into four replicates. 40 μmol / L of the test monomer compound or 5-fluorouracil was added to each well, and the samples were placed in a CO2 incubator and incubated for 48 hours. A blank control group was also included. 10 μL of prepared MTT solution was added to each well, and the samples were placed in a 37°C, 5% CO2 incubator and incubated for another 4 hours. Then, 100 μL of Formazan solution was added, mixed appropriately, shaken several times, and incubated at 37°C for 3–4 hours.

[0039] The absorbance was measured at 570 nm using an ELISA reader, and the absorbance (A) value was recorded. The cell proliferation inhibition rate was then calculated. Cell growth and proliferation inhibition rate (%) = [(Control - Blank) - (Drug-treated - Blank)] / (Control - Blank) × 100%

[0040] The experimental data were statistically analyzed using SPSS 18.0. The values ​​are indicated by the formula. One-way ANOVA was used to compare the means of multiple samples, and the t-test was used to compare the two groups. *P<0.05 and **P<0.01 indicate a significant difference and are statistically significant.

[0041] 1.3 Experimental Results

[0042] 1.3.1 Inhibitory activity of Selaginella monomeric compounds on the proliferation of HepG2 liver cancer cells

[0043] The 24 isolated compounds were used in an inhibitory experiment on the proliferation of HepG2 liver cancer cells. The results showed that at a concentration of 40 μM, compounds 1–24 exhibited varying degrees of inhibitory effects on HepG2 liver cancer cells, with significant differences compared to the blank control group (**P<0.01). Figure 1 .

[0044] 1.3.2 Inhibitory activity of Selaginella monomeric compounds on the proliferation of melanoma A375 cells

[0045] The 24 isolated compounds were used in a melanoma A375 cell proliferation inhibition experiment. The results showed that at a concentration of 40 μM, compounds 1–24 exhibited varying degrees of inhibitory effects on human melanoma A375 cells, with significant differences compared to the blank control group (**P<0.01). Figure 2 .

[0046] 1.3.3 Inhibitory activity of Selaginella monomeric compounds on the proliferation of mouse melanoma B16-F10 cells

[0047] The 24 isolated compounds were used in an inhibitory experiment on the proliferation of mouse melanoma B16-F10 cells. The results showed that at a concentration of 40 μM, compounds 1–24 exhibited varying degrees of inhibitory effects on mouse melanoma B16-F10 cells, with significant differences compared to the blank control group (*P<0.05, **P<0.01). Figure 3 .

[0048] Table 2. Inhibition rate of compound 1-24 on the proliferation of three types of tumor cells (40 μM)

[0049]

[0050] Note: Compared with the control group, *P<0.05, **P<0.01

[0051] Example 2: Anti-triple-negative breast cancer activity

[0052] 1.1 Experimental Materials

[0053] Table 3. Sources of tumor cell lines

[0054]

[0055] Table 4. Names and structures of the tested monomeric compounds (the compound numbers in this example are independent of those in Example 1).

[0056]

[0057]

[0058] All tested monomeric compounds were prepared in the laboratory with a purity of over 98%.

[0059] 1.2 CCK-8 assay to detect the inhibitory activity of Selaginella monomeric compounds on human triple-negative breast cancer MDA-MB-231 and MDA-MB-468 cells.

[0060] Human triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468 were seeded in culture flasks and cultured in DMEM medium (L-15 medium containing 10% fetal bovine serum, 100 units / mL penicillin and 100 μg / mL streptomycin) at 37°C in a 0.1% CO2 incubator.

[0061] Log-phase human triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468 were harvested. The culture medium was discarded, and the cells were washed twice with 2 mL of PBS buffer. The PBS was discarded, and 1 mL of trypsin was added. The cells were gently pipetted, and 2 mL of culture medium was added to stop digestion. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and an appropriate amount of fresh culture medium was added for cell counting. The cells were mixed, and 100 μL of cell suspension was added to each well. Cells were seeded in 96-well plates (1.5 × 10³ cells / well). Different concentrations of the target monomer compound or 5-fluorouracil were added to each well, with four replicates for each drug. The plates were incubated in a CO2 incubator for 48 h, with a blank control group included. After 48 h, CCK-8 reagent was added, and the plates were incubated for another 1.5 h. Cells were then removed, and absorbance (A) was measured at 450 nm using a microplate reader. The cell proliferation inhibition rate was calculated. The inhibition rate at different concentrations was observed, and the IC50 value was determined.

[0062] 1.3 Experimental Results

[0063] Selaginella monomers exhibit inhibitory activity against human triple-negative breast cancer MDA-MB-231 and MDA-MB-468 cells.

[0064] Table 5. Inhibitory activity of compounds against triple-negative breast cancer cells

[0065]

[0066] According to the above IC 50 As can be seen, all compounds of the present invention have certain anti-triple-negative breast cancer activity. Among them, compounds 2, 8, 16 and 17 have better activity, especially compounds 16 (diselaginellins A) and 17 (diselaginellins B), which are similar to the positive control 5-Fu.

[0067] Example 3: Study on the mechanism of action of Diselaginellins B in inhibiting apoptosis in MDA-MB-231 cells.

[0068] For women, breast cancer has become the leading cause of cancer death, posing a serious threat to their physical and mental health. Triple-negative breast cancer is characterized by its high invasiveness, poor prognosis, unclear effective targets, and a trend towards younger onset, making it the subtype with the highest mortality rate among breast cancers. Therefore, finding new therapeutic drugs is of great significance.

[0069] This study, through preliminary in vitro antitumor activity screening of Selaginella tamariscina, found that several alkynylphenol compounds exhibited good inhibitory activity against the proliferation of human triple-negative breast cancer MDA-MB-231 and MDA-MB-468 cells. Among them, the compound diselaginellins B showed the strongest inhibitory effect on the proliferation of human triple-negative breast cancer MDA-MB-231 cells (IC50). 50 Since the concentration was 3.2 μM, this section will use the compound diselaginellins B as the test drug and human triple-negative breast cancer MDA-MB-231 cells as the experimental subject. Annexin V-FITC / PI double staining flow cytometry, Western blot protein blotting, and intracellular reactive oxygen species (ROS) detection will be used to preliminarily explore the mechanism of action of diselaginellins B, a phenolic compound from Selaginella tamariscina, in inducing apoptosis in MDA-MB-231 cells, providing a theoretical basis for subsequent research and development.

[0070] 1.1 Experimental Methods

[0071] 1.1.1 Detection of the effect of diselaginellins B on apoptosis in MDA-MB-231 cells using Annexin V-FITC / PI double staining flow cytometry

[0072] 1×10 5 Human triple-negative breast cancer MDA-MB-231 cells were seeded in 12-well plates and cultured at 37°C in a 0.1% CO2 incubator. Then, 0, 0.5, 1, 2, and 5 μM of the compound diselaginellins B were added, and the cells were cultured for another 36 h. After centrifugation to collect the cells, the Annexin V-FITC / PI apoptosis kit was administered according to the instructions. The cells were washed with PBS, resuspended in Binding Buffer, and collected. 5 μL of Annexin V-FITC was added, followed by 5 μL of PI after 10 min. The cells were incubated at room temperature in the dark for 5 min. Flow cytometry was used to detect apoptosis. The experiment was repeated at least three times.

[0073] 1.1.2 Effects of diselaginellins B on the cell cycle of MDA-MB-231 cells

[0074] Take MDA-MB-231 cells in the logarithmic growth phase, wash twice with 2 mL of PBS buffer, discard the PBS, and prepare a single-cell suspension with culture medium at a cell concentration of 1 × 10⁻⁶ cells / cells. 5 / wells were seeded into 24-well plates. Medium containing 0.5, 2, and 4 μM of diselaginellins B was added, with untreated medium as a blank control. Cells were cultured for 24 h. The medium was aspirated from the wells, and the cells were washed twice with PBS. Fresh medium was added to stop digestion, and the cells were gently pipetted and centrifuged. Cells were collected, washed twice with pre-chilled PBS, and then fixed overnight with pre-chilled 70% ethanol. Fixed cells were collected, centrifuged, and the supernatant was discarded. Cells were washed with pre-chilled PBS, and 100 μL of LNase A and PI (10 μg / mL) were added. Cells were incubated on ice for 30 min, and then analyzed by flow cytometry.

[0075] 1.1.3 Western blot analysis of the expression of diselaginellins B on MDA-MB-231 cell cycle-related proteins

[0076] Log-phase human triple-negative breast cancer MDA-MB-231 cells were harvested, the culture medium was discarded, and the cells were washed twice with 2 mL of PBS buffer. After centrifugation, the supernatant was discarded, and an appropriate amount of fresh culture medium was added for cell counting. The cells were then mixed and seeded into 6-well plates. Different concentrations (0, 0.1, 0.25, 0.5, and 1 μM) of the compound diselaginellins B were added. After 24 h of drug administration, the cells were washed twice with PBS, and cell lysis buffer was added. The cells were lysed on ice for 1.5 h, centrifuged at 4 °C for 10 min, and the supernatant was collected as total cell protein. Standard protein solutions of different concentrations were prepared and added to the standard wells of 96-well plates. An appropriate volume of sample was then added to the sample plate of the 96-well plate, and G250 staining solution was added to each well. After incubation for 5 min, the protein content of the sample was determined by measuring the absorbance using a microplate reader. 30 μg of total protein from each group was subjected to SDS-polyacrylamide gel electrophoresis (PAGE). The protein was then transferred to a polyvinylidene fluoride (PVDF) membrane using a Trans-Blot semi-dry transfer system. The membrane was blocked with 5% skim milk powder at 4°C for 2 h to block the monoclonal antibodies against pCHK1, CHK1, pCdc25c, Cdc25c, pCdc2, and Cyclin B1. The membrane was incubated overnight, followed by incubation with the corresponding secondary antibody IgG (1:2000) at room temperature for 2 h. The membrane was rinsed with deionized water, photographed using a gel imaging system, and the scanned images were analyzed using a quantitative analysis system.

[0077] 1.1.4 Regulation of intracellular ROS by diselaginellins B in MDA-MB-231 cells

[0078] Cells in logarithmic growth phase were fed at a rate of 1×10⁻⁶. 5The cells were seeded at a density of 1 / 2 wells in 12-well plates and incubated at 37°C in a 0.1% CO2 incubator. Then, 0, 0.5, 2, and 5 μM of the compound diselaginellins B were added, and the cells were incubated for another 48 h. Next, 10 mM of the DCFH-DA probe was added, and the cells were co-incubated at 37°C for 30 min. Residual fluorescent probes were removed using pre-cooled PBS, and the cells were washed twice. The MDA-MB-231 cells were then analyzed by flow cytometry.

[0079] 1.2 Experimental Results

[0080] 1.2.1 Detection of the effect of diselaginellins B on apoptosis in MDA-MB-231 cells using Annexin V-FITC / PI double staining flow cytometry

[0081] Flow cytometry results showed that the apoptosis rate of human triple-negative breast cancer MDA-MB-231 cells in the blank control group was 0.87%. After treating MDA-MB-231 cells with 0.5, 1, 2, and 5 μM diselaginellins B for 36 h, the apoptosis rate significantly increased with increasing drug concentration, exhibiting a dose-dependent effect. Furthermore, the differences between different drug concentrations and the blank control group were statistically significant (**P<0.01). This indicates that diselaginellins B can induce apoptosis in human triple-negative breast cancer MDA-MB-231 cells in a dose-dependent manner. (See details...) Figure 4 .

[0082] 1.2.2 Effects of diselaginellins B on the cell cycle of MDA-MB-231 cells

[0083] As the concentration of diselaginellins B increased, the flow cytometry results were as follows: Figure 5 As shown, when MDA-MB-231 cells were treated with different concentrations (0.5, 2, and 4 μM) of the compound diselaginellins B for 24 h, compared with the blank control group, the proportion of cells in the G2 / M phase significantly increased, from 9.73% to 15.67%, 16.84%, and 19.77%, respectively; the corresponding proportion of cells in the G0 / G1 phase decreased, from 84.21% in the control group to 71.67%, 68.03%, and 56.90%, respectively; no significant change was observed in the S phase cells. These results suggest that diselaginellins B can arrest the MDA-MB-231 cell cycle in the G2 / M phase.

[0084] 1.2.3 Effect of diselaginellins B on the expression levels of MDA-MB-231 cell cycle-related proteins

[0085] One of the main molecular mechanisms by which intracellular and extracellular DNA damage signals lead to G2 / M phase arrest of the cell cycle is through the regulation of the Chk1-Cdc25C-Cyclin B1 pathway, thereby inhibiting the activity of the Cdc2-Cyclin B1 complex and its entry into the nucleus, ultimately causing G2 / M phase arrest. When cells detect DNA damage, they arrest in the G2 phase for examination. Chk1 in the nucleolus is phosphorylated, and the phosphorylated Chk1 is activated, leading to the phosphorylation of Cdc25c. Cdc25c is then phosphorylated to form pCdc25c, which is subsequently secreted into the cytoplasm. Thus, Cdc25c, which would normally reside in the nucleus, cannot perform its function of dephosphorylating Cdc2 [pcdc2(Tyr15)]. Dephosphorylation of cdc2 (i.e., reducing pCdc2 expression) is key to the activation of the Cdc2-Cyclin B1 complex, which in turn is a prerequisite for cells to transition from G2 to M phase. Therefore, Western blot data showed that after 24 hours of administration of different concentrations (0.1, 0.25, 0.5, and 1 μM) of the compound diselaginellins B, the phosphorylation level of pChk1 increased with increasing concentration (P < 0.05), while pCdc25c also increased with increasing concentration (P < 0.001). This indicates that with increasing drug concentration, phosphorylated Chk1 is activated, further phosphorylating Cdc25c, causing Cdc25c to be secreted from the nucleus and lose its function of dephosphorylating Cdc2. Conversely, pCdc2 expression increases with increasing drug concentration (P < 0.05), while Cyclin B1 expression decreases with increasing drug concentration (P < 0.05). This keeps the Cdc2-Cyclin B1 complex in an inactive state, preventing cells from entering the M phase. Figure 6 , 7 .

[0086] 1.2.4 Effects of diselaginellins B on intracellular ROS in MDA-MB-231 cells

[0087] Reactive oxygen species (ROS) are a class of reactive oxygen species produced during aerobic metabolism. Excessive production can attack biomolecules and cell walls, leading to apoptosis and systemic damage. Therefore, elevated intracellular ROS levels are considered a key factor in initiating the apoptosis process. This experiment used the fluorescent probe DCF to treat cells, and then used flow cytometry to detect ROS levels in MDA-MB-231 cells treated with different concentrations of diselaginellins B. Figure 8As shown, compared with the control group, the curve of the drug-treated group shifted significantly to the right with the increase of drug concentration (0.5, 2, 5 μM), indicating that the increase of compound concentration can promote the production of intracellular reactive oxygen species, significantly increase the level of intracellular ROS, and thus induce apoptosis of MDA-MB-231 cells.

[0088] Continuous growth signal stimulation, evasion of growth inhibition, and resistance to programmed cell death are all important biological characteristics of tumor cells. The imbalance between cell proliferation and apoptosis is an important factor in the occurrence and development of tumors.

[0089] The results showed that diselaginellins B had good inhibitory activity on the proliferation of triple-negative breast cancer MDA-MB-231 cells. Therefore, this chapter conducted experiments on the molecular mechanism of action of this compound. Flow cytometry results showed that diselaginellins B could significantly inhibit the proliferation of MDA-MB-231 cells and induce apoptosis in a significant dose-dependent manner. Further studies showed that diselaginellins B could increase the percentage of MDA-MB-231 cells in the G2 / M phase, which is speculated to be due to the induction of apoptosis by G2 / M phase arrest in MDA-MB-231 cells.

[0090] Subsequently, Western blot analysis was used to detect the expression of cell cycle-related proteins in MDA-MB-231 cells by diselaginellins B. The results showed that the expression of pChk1, pCdc25c, and pCdc2 proteins increased, while the expression of Cyclin B1 decreased. This suggests that diselaginellins B can activate the Chk1-Cdc25C-Cyclin B1 signaling pathway, inhibit the activity of the Cdc2-Cyclin B1 complex and its entry into the nucleus, ultimately causing G2 / M phase arrest. At the same time, diselaginellins B can significantly increase the intracellular reactive oxygen species (ROS) content and induce apoptosis.

[0091] In summary, diselaginellins B significantly promotes apoptosis in triple-negative breast cancer MDA-MB-231 cells, inducing G2 / M phase arrest. Furthermore, the molecular mechanism of diselaginellins B's anti-triple-negative breast cancer effect was explored from the perspective of regulating cell cycle-related protein expression, providing an experimental basis for its application in the prevention and treatment of triple-negative breast cancer.

Claims

1. Use of one of the following compounds or a combination of two in the preparation of an anti-melanoma or breast cancer drug; the compound is diselaginellins A, diselaginellins B; 。 2. Use according to claim 1, characterized in that, the drug is a drug that promotes apoptosis of cancer cells.

3. Use according to claim 1, characterized in that, the drug is a drug that causes G2 / M phase arrest of cancer cells.

4. Use according to claim 1, characterized in that, the drug is a drug that increases the content of reactive oxygen species in cancer cells.

5. Use according to claim 1, characterized in that, the breast cancer is triple negative breast cancer.

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