Use of a huperzia serrata extract
By using the extract of Chinese fern ent-8(14),15-pinediene-2β,19-diol, the proliferation and migration of TNBC cells were inhibited, which solved the problems of target deficiency and side effects of existing TNBC treatments and provided a new treatment method.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ANHUI UNIVERSITY OF TECHNOLOGY
- Filing Date
- 2024-01-25
- Publication Date
- 2026-07-14
Smart Images
Figure CN117883420B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of an extract of small-leaved Chinese fern. Background Technology
[0002] ent-8(14),15-pinediene-2β,19-diol (JXE-23) is a compound isolated from the small-leaved Chinese fern. Previous studies have found that JXE-23, together with other diterpenes, can significantly reduce the amount of NO produced in cells by blocking the expression and transcription of iNOS protein and inflammatory mediators TNF-α and IL-6, thus exerting an anti-inflammatory effect.
[0003] Triple-negative breast cancer (TNBC) is a specific subtype of breast cancer, known for its aggressiveness, poor prognosis, limited clinical treatment options, and lack of effective specific targeted therapies. TNBC accounts for 15%-20% of diagnosed breast cancers and is characterized by the absence of three specific receptors: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Treating this aggressive disease is challenging due to the lack of targeted receptors expressed in other breast cancer types and its complex pathological processes. To date, non-specific treatments such as surgery, conventional radiotherapy, and chemotherapy have been the only treatment options. Therefore, developing new TNBC treatments is crucial. Individual differences among TNBC patients are significant, clinical data are limited, and most TNBC subtyping is based on the mRNA levels of different genes. However, because proteins undergo multiple modifications and regulatory steps during translation, mRNA expression levels do not accurately reflect protein expression levels. This deficiency significantly impacts targeted therapy and postoperative prediction. Currently, there are no effective targeted therapies for TNBC, and multiple treatment approaches may be required to significantly improve patient prognosis. Chemotherapy is one of the main treatments for TNBC, and it is often used in combination with other drugs in clinical practice. The combination chemotherapy of taxanes and anthracyclines is the standard treatment for early-stage TNBC patients, but it increases the risk of cardiac death, secondary leukemia, and myelodysplastic syndromes to some extent. Carboplatin increases the incidence of neutropenia and thrombocytopenia, while bevacizumab can cause hypertension, infection, thrombosis, bleeding, and postoperative complications. To develop new drugs for the treatment of TNBC, this invention provides the following technical solutions. Summary of the Invention
[0004] The purpose of this invention is to provide an application of the extract of *Pteris vittata*, offering a new approach and method for the clinical treatment of TNBC.
[0005] The objective of this invention can be achieved through the following technical solutions:
[0006] The use of a small-leaved Chinese fern extract as the sole active ingredient in the preparation of a drug for the treatment and / or prevention of triple-negative breast cancer;
[0007] The extract of *Pteris vittata* is ent-8(14),15-pinediene-2β,19-diol:
[0008] Furthermore, the drug is an inhibitor that suppresses the proliferation of cancer cells.
[0009] Furthermore, the drug is an inhibitor that inhibits the migration of cancer cells.
[0010] Furthermore, the drug is an inhibitor of EGFR / Akt / ERK / p70. S6K An inhibitor of the signaling pathway that reduces the viability of MDA-MB-468 cells.
[0011] The beneficial effects of this invention are:
[0012] The compound ent-8(14),15-pinediene-2β,19-diol described in this invention was isolated from *Pteris vittata* and significantly inhibited the proliferation of breast cancer cells. Scratch assays showed that this compound inhibited the migration of highly migratory TNBC cells. Western blot experiments further verified that this compound inhibits the proliferation of EGFR / Akt / ERK / p70 cells. S6K The signaling pathway reduced the viability of MDA-MB-468 cells. These results indicate that the ent-8(14),15-pinene-2β,19-diol described in this invention possesses good anti-TNBC activity and has potential application value in the future treatment of TNBC. Attached Figure Description
[0013] The invention will now be further described with reference to the accompanying drawings.
[0014] Figure 1 This is a schematic diagram of the MTT assay used to detect the inhibition of TNBC cell growth by the compound ent-8(14),15-pinediene-2β,19-diol.
[0015] Figure 2 This is a schematic diagram of the effect of compound ent-8(14),15-pinediene-2β,19-diol on the migration of MDA-MB-468 cells in TNBC cells (scale bar: 100 μm, ***p<0.001vs Con);
[0016] Figure 3This is a schematic diagram showing the effect of compound ent-8(14),15-pinediene-2β,19-diol on the morphology of TNBC cells MDA-MB-468.
[0017] Figure 4 The compound ent-8(14),15-pinediene-2β,19-diol is associated with EGFR and its downstream signaling pathway-related proteins Akt, ERK, and p70. S6K A diagram illustrating the impact of expression. Detailed Implementation
[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0019] ent-8(14),15-pinediene-2β,19-diol is a compound isolated from *Pteris vittata*, and its structural formula is as follows:
[0020]
[0021] Example 1: MTT assay to determine the inhibitory effect of ent-8(14),15-pinediene-2β,19-diol on cancer cell growth.
[0022] (1) Inoculating cells
[0023] Cells with good growth and a survival rate of 90% were passaged, cell pellets were collected, and single-cell suspensions were prepared. After cell counting, cells were seeded into 96-well plates at an appropriate cell density (1000-10000 cells / well), with 100 μL of single-cell suspension added to each well. Three replicates were set up for each group. After observing the cell status under a microscope, the plates were placed in an incubator containing 5% CO2 and cultured at 37°C, 5% CO2, and saturated humidity for 24 hours.
[0024] (2) Chemical treatment
[0025] After the cells adhered for 24 hours, an appropriate drug concentration gradient was set according to the cells' sensitivity to the drug. Different concentrations of drug culture medium were prepared according to the drug formulation. The 96-well plate was taken out, the supernatant was discarded, and different concentrations of drug culture medium were added in sequence. Three replicates were set up for each group, and a blank control was set up. After observing the cell status under a microscope, the cells were placed in a CO2 incubator for further culture.
[0026] (3) MTT test
[0027] After 70 hours of drug incubation, 10 μL of 5 mg / mL MTT solution was added to each well. After observing the cell status under a microscope, the cells were incubated in a CO2 incubator for another 2 hours. When the MTT in the control group had fully bound to the cells to form insoluble blue-purple crystals called formazan, the 96-well plate was removed, the solution in each well was carefully discarded, and 150 μL of DMSO was added. The plate was shaken at low speed for 5 minutes to allow the DMSO to fully dissolve the formazan in the cells. The microplate reader was turned on, the wavelength was set to 490 nm, the absorbance (OD value) was read, and the data was saved. Each experiment was repeated three times independently.
[0028] (4) Data Analysis
[0029] The inhibition rate of cell growth at different drug concentrations was calculated using the formula: Inhibition rate (%) = (OD value of control group - OD value of experimental group) / (OD value of control group - OD value of blank group) × 100. The half-inhibitory concentration (IC50) of the drug in the cells was calculated using GraphPad Prism 8 software.
[0030] The MTT assay was used to screen ent-8(14),15-pinediene-2β,19-diol, and the results are shown in Table 1. In TNBC cells, ent-8(14),15-pinediene-2β,19-diol showed the lowest half-maximal inhibitory concentration (IC50) against MDA-MB-468.
[0031] To further verify the inhibitory effect of ent-8(14),15-piperene-2β,19-diol on the growth of MDA-MB-468 cells, the MTT assay was used to detect the inhibition rate of ent-8(14),15-piperene-2β,19-diol on MDA-MB-468 cells at 24h, 48h, and 72h. The results are as follows: Figure 1 As shown, ent-8(14),15-pinene-2β,19-diol showed dose-dependent inhibition of MDA-MB-468.
[0032] Table 1 IC50 values (μM±SD) of ent-8(14),15-pinediene-2β,19-diol for different TNBC cells
[0033]
[0034] Example 2: Effect of compound ent-8(14),15-pinene-2β,19-diol on the proliferation of MDA-MB-468 cells by trypan blue staining and counting method
[0035] (1) Inoculating cells
[0036] Cells in good growth condition with a cell density of 90% were passaged, centrifuged at 800 rpm for 5 min, and the resulting cell pellet was resuspended in 1 mL of culture medium to prepare a single-cell suspension. After cell counting, the suspension was prepared at a concentration of 3.5 × 10⁻⁶ cells / mL. 5 Cells were seeded into 12-well plates at a density of cells per well. After observing the cell condition under a microscope, the plates were placed in an incubator containing 5% CO2 and cultured at 37°C, 5% CO2, and saturated humidity for 24 hours.
[0037] (2) Chemical treatment
[0038] After cells adhered for 24 hours, appropriate drug concentration gradients of 2.5 μM, 5 μM, and 10 μM were set up according to the half-maximal inhibitory concentration (IC50) of the drug. Different concentrations of drug culture medium were prepared. The 12-well plates were removed, the supernatant was discarded, and different concentrations of drug culture medium were added in sequence. Three replicates were set up for each concentration, and a blank control was set up. After observing the cell status under a microscope, the cells were continued to be cultured in a CO2 incubator.
[0039] (3) Trypan blue staining count detection
[0040] After incubation with the drug for 24h, 48h, and 72h, cells of a concentration gradient were digested and resuspended. Each concentration of cell suspension was diluted with PBS in triplicate. After staining with 0.4% trypan blue for 2min, cells were counted under an inverted microscope using a hemocytometer. The counts were recorded at 24h, 48h, and 72h.
[0041] (4) Data Analysis
[0042] Each concentration was counted independently three times. The results were plotted and statistically analyzed using GraphPad Prism 8.0.2 software to obtain the cell growth curve after drug treatment.
[0043] Example 3: Effect of compound ent-8(14),15-pinediene-2β,19-diol on the migration of MDA-MB-468 cells using scratch assay
[0044] (1) Inoculating cells
[0045] Beforehand, draw three horizontal lines on the bottom of the 6-well plate with a marker to ensure consistent photo positions. Passage cells in the logarithmic growth phase, collect the cell pellet, prepare a single-cell suspension, count the cells, and then arrange them at an appropriate cell density (MDA-MB-468: 5.0 × 10⁻⁶). 5 Units / well; MDA-MB-468: 1.0 × 10 6 (Cells / well) were seeded onto plates, and 2 mL of single-cell suspension was added to each well. After mixing using the "cross-hatching" method, the cell state was observed under a microscope. The cells were cultured overnight at 37°C, 5% CO2, and saturated humidity.
[0046] (2) Drawing lines
[0047] The following day, under the microscope, cells were observed to cover the entire bottom of the dish (ideally a 100% confluent monolayer). A 200μL pipette tip was used for streaking, maintaining the streaking path perpendicular to the three horizontal lines. The streaking was performed along a sterile ruler from one end to the other in a "|" shape. Clear streaks were visible on the cell surface. The old culture medium was discarded, and the cells were washed several times with PBS to remove any cells detached during the streaking process. The cell condition was observed under the microscope, and photographs were taken and saved as a 0h control group. The photographed area (the area above or below the intersection of the streak and the positioning lines) was recorded for future observation. Note: When streaking, the pipette tip must always be perpendicular to the cell surface and the same tip must be used.
[0048] (3) Chemical treatment
[0049] To reduce false positive results caused by cell proliferation and minimize its impact on experimental results, appropriate concentrations of the drug (MDA-MB-468: 0 μM, 2.5 μM, 5 μM, 10 μM) were prepared in low serum medium (2% FBS). After 72 hours, the differences in migration ability between the treatment group and the control group were observed and photographed.
[0050] (4) Observation and photography under an optical microscope
[0051] After 72 hours, observe the healing of the scratches, discard the old culture medium, wash three times with PBS, find the marked position of the 0h control group and take a picture to record it, and save the picture as a Tif format.
[0052] (5) Data Analysis
[0053] The scratch area was calculated using ImageJ software. Cell migration rate (%) = (area of control group - area of experimental group / area of control group) × 100. Each experiment was independently repeated 3 times. The results were plotted and statistically analyzed using GraphPad Prism 8 software.
[0054] like Figure 2 As shown, the scratch width in the control group gradually narrowed over time, while in the 72-hour condition, the drug-treated group significantly inhibited scratch healing with increasing concentration, exhibiting a dose-dependent effect. These experimental results indicate that the compound ent-8(14),15-pinene-2β,19-diol can significantly inhibit the migration of MDA-MB-468 cells.
[0055] Example 4: Phase contrast microscopy imaging to detect the effect of compound ent-8(14),15-pinene-2β,19-diol on morphological changes in MDA-MB-468 cells
[0056] (1) Inoculating cells
[0057] Cells in good growth condition with a cell density of 90% were passaged, centrifuged at 800 rpm for 5 min, and the resulting cell pellet was resuspended in 1 mL of culture medium to prepare a single-cell suspension. After cell counting, the suspension was prepared at a concentration of 1.0 × 10⁻⁶ cells / mL. 5 Cells were seeded into 6-well plates at a density of cells per well. After observing the cell condition under a microscope, the plates were placed in an incubator containing 5% CO2 and cultured at 37°C, 5% CO2, and saturated humidity for 24 hours.
[0058] (2) Chemical treatment
[0059] After the cells adhered for 24 hours, drug culture media with drug concentration gradients of 2.5 μM, 5 μM and 10 μM were prepared. The 6-well plates were removed, the supernatant was discarded, and the drug culture media of different concentrations were added in sequence. A control group without drug was set up. After observing the cell status under a microscope, the cells were placed in a CO2 incubator for another 24 hours.
[0060] (3) Cell morphology observation
[0061] like Figure 3 As shown, after 24 hours of cell treatment, the 6-well plates were removed, and the cell morphology was observed and photographed under an inverted phase-contrast microscope to obtain cell phase-contrast morphology images. Then, the 6-well plates were washed once with PBS, fixed with 1 mL of methanol for 15 min, washed again with PBS, and stained with 0.005% crystal violet for 20 min. Cells at each concentration were photographed under a regular inverted microscope to obtain crystal violet stained cell morphology images.
[0062] Example 5: Western blot analysis of the effect of compound ent-8(14),15-pinene-2β,19-diol on protein expression in MDA-MB-468 cells
[0063] (1) Inoculating cells
[0064] MDA-MB-468 cells with good growth and 90% confluence were passaged, and the cell pellet was collected and prepared into a single-cell suspension. After cell counting, the suspension was prepared at a concentration of 1.0 × 10⁻⁶ cells / cells. 5 Cells were seeded into 6-well plates at a density of 2 mL of single-cell suspension per well. After mixing with the "cross-shading" method, the cells were observed under a microscope and cultured at 37°C, 5% CO2, and saturated humidity for 24 h.
[0065] (2) Chemical treatment
[0066] After the cells adhered for 24 hours, drug culture media with drug concentration gradients of 0 μM, 2.5 μM, 5 μM and 10 μM were prepared. The 6-well plates were removed, the supernatant was discarded, and the drug culture media of different concentrations were added in sequence. The plates were then placed in a CO2 incubator for another 24 hours.
[0067] (3) Cell protein extraction
[0068] The state of MDA-MB-468 cells after drug treatment was observed under a microscope, and the content of cell lysis buffer was adjusted according to the cell density in each well. The 6-well plate was placed on ice, and the entire process of protein collection was carried out on ice. The supernatant was discarded, and the cells were washed with pre-chilled PBS. The PBS was discarded, and then the appropriate amount of cell lysis buffer was added evenly. After complete lysis, the lysed cells were collected with a pre-chilled, clean cell scraper into pre-chilled 1.5 mL centrifuge tubes. The tubes were boiled twice in a 100°C water bath for 4 min each time, with a total time not exceeding 10 min, to ensure complete protein denaturation. After vortexing, the tubes were stored at -20°C.
[0069] (4) Western blot detection of proteins
[0070] Protein samples were loaded into each well of a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and separated by electrophoresis, then transferred to an NC membrane. After blocking with 5% (w / v) skim milk at room temperature for 1 h, the NC membrane containing the protein was incubated with the corresponding protein's primary antibody and slowly incubated overnight at 4°C using a rotary shaker. After washing four times with TBS-T containing 0.05% (v / v) Tween-20, the membrane was incubated with a specific secondary antibody at room temperature for 2 h. Then, the membrane was washed four more times with TBS-T. The immunoreaction bands were developed using an enhanced chemiluminescent substrate. Anti-β-actin antibody was used as a control. Finally, the bands were straightened and trimmed using PS software. The results are shown below. Figure 4 As shown, ImageJ software was used to perform quantitative analysis on the grayscale values of the strips, and Excel software was used to normalize them.
[0071] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.
Claims
1. The use of a small-leaved Chinese fern extract as the sole active ingredient in the preparation of a medicament for the treatment and / or prevention of triple-negative breast cancer; The extract of *Pteris vittata* is... ent- 8(14),15-Pinocene-2 β ,19-diol, with the following structural formula: 。