Application of mangnolia officinalis extract in preparation of antitumor drugs

Abstract

The invention provides application of a mangnolia officinalis extract serving as a medicinal active component to preparation of an anti-tumor drug. According to the present invention, the magnolia officinalis extract tetrahydromagnolol has strong anti-cancer activity, anti-oxidation and anti-aging effects on lung cancer, intestinal cancer, pancreatic cancer and other malignant tumor cells, and the tetrahydromagnolol has strong proliferation inhibition activity on malignant tumor cells so as to effectively provide the anti-tumor effect; therefore, the mangnolia officinalis extract can be used for preparing antitumor drugs.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to use of a magnolia bark extract in preparing an anti-tumor drug. Background Art

[0002] Magnolia officinalis is the dried bark, root bark and branch bark of Magnolia officinalis, a plant of the Magnoliaceae family. The Chinese Pharmacopoeia records that it is bitter, pungent, warm, and enters the spleen, stomach, lung, and large intestine meridians, and has the effects of drying dampness and eliminating phlegm, and removing qi and fullness. Magnolia officinalis contains a wealth of medicinal active ingredients, including magnolol, honokiol, 4-O-methylhonokiol, and magnolol. Tetrahydromagnokiol is the main metabolite of magnolol, and its efficacy is better than that of magnolol.

[0003] Chinese patent literature discloses "Honokiol derivatives, preparation methods thereof and use thereof in the preparation of anti-tumor drugs", and its publication number is CN 103553888A. This patent obtains honokiol derivatives with a new skeleton by extracting and isolating them from the ripe fruits of Magnolia grandiflora by adopting an activity tracking separation method, and proves that the honokiol derivatives have a significant effect of inhibiting the proliferation of tumor cells; the honokiol derivatives have a strong ability to inhibit the anti-apoptotic factor Bcl-2 of tumor cells and enhance the activity of Caspase3.

[0004] So far, there has been no report on the application of tetrahydromagnolia bark ol in the treatment of malignant tumors. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a use of a Magnolia bark extract for preparing an anti-tumor drug, wherein the Magnolia bark extract has a strong proliferation inhibitory activity on malignant tumor cells, thereby effectively exerting an anti-tumor effect.

[0006] To achieve the above-mentioned object and other related objects, the present invention provides a use of a Magnolia bark extract as a medicinal active ingredient for preparing an anti-tumor drug.

[0007] Preferably, the Magnolia bark extract is tetrahydromagnolia bark extract.

[0008] Preferably, the anti-tumor drug is selected from one or more of anti-lung cancer drugs, anti-colon cancer drugs and anti-pancreatic cancer drugs.

[0009] The present invention also provides an anti-lung cancer drug, which contains 30-90 μM tetrahydromagnolia nobile.

[0010] Preferably, the anti-lung cancer drug is used to inhibit the proliferation ability of lung cancer A549 cells and lung cancer H1299 cells.

[0011] The present invention also provides an anti-intestinal cancer drug, which contains 25-75 μM tetrahydromagnolia nobile.

[0012] Preferably, the anti-colon cancer drug is used to inhibit the proliferation ability of colon cancer HT29 cells and colon cancer HCT116 cells.

[0013] The present invention also provides an anti-pancreatic cancer drug, which contains 40-80 μM tetrahydromagnolia nobile.

[0014] Preferably, the anti-pancreatic cancer drug is used to inhibit the proliferation ability of pancreatic cancer SW1990 cells and PANC-1 cells.

[0015] As described above, the present invention has the following beneficial effects: the magnolia bark extract tetrahydromagnolia ol has strong anti-cancer activity and antioxidant and anti-aging effects on malignant tumor cells such as lung cancer, intestinal cancer, pancreatic cancer, etc., and tetrahydromagnolia ol has strong proliferation inhibition activity on malignant tumor cells, thereby effectively exerting an anti-tumor effect; therefore, the magnolia bark extract can be used to prepare anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown is a curve chart showing the effect of tetrahydromagnolia bark ol on the proliferation of human embryonic lung fibroblast cell line HELF, human normal intestinal epithelial cell line NCM460, and human normal pancreatic ductal epithelial cell line H6C7.

[0017] Figure 2 Shown is a curve chart of the effect of tetrahydromagnolia bark ol on lung cancer, intestinal cancer and pancreatic cell proliferation.

[0018] Figure 3 Shown is the effect of different concentrations of tetrahydromagnolia nobile (THM) on lung cancer cell apoptosis and quantitative analysis.

[0019] Figure 4 Shown is the effect of different concentrations of tetrahydromagnolia nobile (THM) on apoptosis of colorectal cancer cells and quantitative analysis.

[0020] Figure 5 Shown is the effect of different concentrations of tetrahydromagnolia nobile (THM) on pancreatic cancer cell apoptosis and quantitative analysis.

[0021] Figure 6 Shown is the effect of different doses of tetrahydromagnolia nobile on the clone-forming ability of lung cancer A549 and H1299 cells.

[0022] Figure 7 Shown is the effect of different doses of tetrahydromagnolia nobile on the clone-forming ability of colorectal cancer HT29 and HCT116 cells.

[0023] Figure 8Shown are the effects of different doses of tetrahydromagnolia nobile on the clone-forming ability of pancreatic cancer SW1990 and PANC-1 cells. DETAILED DESCRIPTION

[0024] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0025] It should be noted that the process equipment or devices not specifically specified in the following embodiments are all conventional equipment or devices in the art.

[0026] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0027] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0028] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0029] The present application embodiment provides a use of a magnolia bark extract as a medicinal active ingredient for preparing an anti-tumor drug, wherein the magnolia bark extract is tetrahydromagnolin. The anti-tumor drug is selected from one or more of an anti-lung cancer drug, an anti-intestinal cancer drug, and an anti-pancreatic cancer drug.

[0030] The anti-lung cancer drug contains 30-90 μM tetrahydromagnolia nobile and is used to inhibit the proliferation ability of lung cancer A549 cells and lung cancer H1299 cells.

[0031] The anti-intestinal cancer drug contains 25-75 μM tetrahydromagnolia nobile and is used to inhibit the proliferation ability of intestinal cancer HT29 cells and intestinal cancer HCT116 cells.

[0032] The anti-pancreatic cancer drug contains 40-80 μM tetrahydromagnolia nobile and is used to inhibit the proliferation ability of pancreatic cancer SW1990 cells and PANC-1 cells.

[0033] In the following examples of this application, the sources of the reagents used are as follows: Tetrahydromagnolia nobile (Shanghai Yuanye Biotechnology Co., Ltd.); 10% fetal bovine serum (Vistec, SA); 1640, IMDM, DMEM culture medium (Shanghai Yuanpei Biotechnology Co., Ltd.); 0.25% trypsin (Shanghai Yuanpei Biotechnology Co., Ltd.); CCK8 kit (Dalian Meilun Biotechnology Co., Ltd.); crystal violet stain (Beyotime); cell apoptosis detection kit (Shanghai Yisheng Biotechnology) In the following examples of this application, the cell lines used are as follows: Human embryonic lung fibroblast cell line HELF, lung cancer A549 cells, lung cancer H1299 cells; human normal intestinal epithelial cell line NCM460, colorectal cancer HT29 cells, colorectal cancer HCT116 cells; human normal pancreatic ductal epithelial cell line H6C7, pancreatic cancer SW1990 colorectal cancer, pancreatic cancer PANC-1 colorectal cancer were all purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0034] Cell Counting Kit 8 (CCK8 method) detects cell proliferation, including the following steps: (1) Human embryonic lung fibroblasts HELF, lung cancer A549 cells and lung cancer H1299 cells with good cell growth status and in the logarithmic growth phase, human normal intestinal epithelial cells NCM460, intestinal cancer HT29, intestinal cancer HCT116 cells, human normal pancreatic ductal epithelial cells H6C7, pancreatic cancer SW1990 cells, and pancreatic cancer PANC-1 cells were selected for digestion with trypsin, and the cells were terminated by adding complete culture medium containing 10% FBS. After being pipetted into a cell suspension, the cell suspension was transferred to an EP tube, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and complete culture medium was added to make a cell suspension; (2) Count the cells using a cell counting plate. Inoculate 5,000 cells in 96-well plates with 6 replicate wells per group of cells at a volume of 100 μL per well. Add an equal amount of PBS solution to the wells on the edges to prevent edge effects. (3) Incubate the cells overnight in a 37°C, 5% CO2 incubator with conventional culture medium. Aspirate the liquid culture medium in each well and add prepared tetrahydromagnolia nobile culture medium with different concentration gradients (10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM) to each well of different cells and continue culturing. (4) After 24 hours of incubation, remove the culture medium from the plate in a dark environment, prepare the CCK8 solution required for the experiment, add 100 μL of solution to each well (prevent bubbles from forming during the addition process to avoid inaccurate measurement by the microplate reader), and incubate in the incubator for 1-2 hours; (5) Adjust the wavelength of the automatic microplate reader to 450 nm and measure the OD value of each well; (6) The growth curve of each group of cells was drawn according to the OD value, and the cell proliferation was calculated and analyzed using Graphpad 9.5. The results are shown in the figure. Figure 1 and Figure 2 shown.

[0035] like Figure 1 and Figure 2 As shown in the figure, the CCK8 method was used to detect the effects of different concentrations on the growth and proliferation of human embryonic lung fibroblast cell line HELF, lung cancer A549, H1299, human normal intestinal epithelial cells NCM460, intestinal cancer HT29, HCT116, human normal pancreatic ductal epithelial cells H6C7, pancreatic cancer SW1990, and PANC-1. Tetrahydromagnolia phenol at each experimental concentration had a certain inhibitory effect on the growth and proliferation of lung cancer, intestinal cancer, and pancreatic cancer cells, and the cell growth inhibition rate gradually increased with the increase of dose, with obvious dose dependence. Compared with cancer cells, normal human tissue cells (human embryonic lung fibroblast cell line HELF, human normal intestinal epithelial cell line NCM460, and human normal pancreatic ductal epithelial cell line H6C7) were less sensitive to the drug effects of tetrahydromagnolia phenol, indicating that tetrahydromagnolia phenol can specifically kill cancer cells.

[0036] The cell apoptosis experiment includes the following steps: (1) Select lung cancer cells A549 and H1299, intestinal cancer cells HT29 and HCT116, and pancreatic cancer cells SW1990 and PANC-1 that are in good growth condition and in the logarithmic growth phase, digest them with trypsin, add complete culture medium containing 10% FBS to terminate the digestion, centrifuge, resuspend, and count; (2) Inoculate cells into a 6-well plate at a density of 3 × 105 cells / well, shake gently, and then culture in an incubator overnight. When the cells adhere to the wall and the density reaches about 70%-80%, discard the culture medium, wash the cells with PBS, add different concentrations of tetrahydromagnolia bark ol, and then culture in an incubator for 24 hours in the dark. The concentration of tetrahydromagnolia bark ol is as follows: Figure 3 , Figure 4 and Figure 5 As shown; (3) Collect the cell supernatant and culture it in a labeled 15 ml centrifuge tube, wash with PBS, digest with trypsin, stop digestion with complete medium, blow the cells slowly and gently to minimize mechanical death, and transfer all the cell suspension to the 15 ml centrifuge tube containing the collected supernatant; (4) Centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells with 500 μL PBS and collect them in a 1.5 mL EP tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant; (5) Add 200 μL of 1× binding buffer to each EP tube containing cell pellets to resuspend and mix thoroughly. Then add 3 μL of Annexin V-FITC and 3 μL of PI dye solution respectively. After mixing thoroughly, transfer to a flow tube and incubate in the dark at room temperature for 15 min. (6) Gently blow the liquid through the pipette, put it on the machine, and use the flow cytometer to analyze the cell staining and count the cell apoptosis. The results are as follows Figure 3 , Figure 4 and Figure 5 shown.

[0037] like Figure 3 , Figure 4 and Figure 5 As shown, the cell apoptosis experiment was used to detect the effect of different concentrations of tetrahydromagnolia phenol on the growth and proliferation of lung cancer, intestinal cancer and pancreatic cancer cells. According to the experimental data, different concentrations of tetrahydromagnolia phenol were used to treat different cancer cells. As the drug concentration increased, the apoptosis rate of cells treated with tetrahydromagnolia phenol increased significantly compared with the control group, showing a dose-dependent manner.

[0038] The plate monoclonal formation experiment includes the following steps: (1) Lung cancer cells A549 and H1299, intestinal cancer cells HT29 and HCT116, and pancreatic cancer cells SW1990 and PANC-1 in the logarithmic growth phase, with good growth status and moderate density were selected for digestion, centrifugation, resuspending, and counting; (2) Inoculate 5000 cells / dish into a 6 cm dish, shake gently and place in an incubator overnight. After the cells attach to the wall and grow into a normal morphology, discard the culture medium and add medium containing or without drugs to the culture dish according to the drug concentration gradient (this operation is protected from light). After 24 hours, replace with fresh medium. The concentration of tetrahydromagnolia bark ol is as follows: Figure 6 , Figure 7 and Figure 8 As shown; (3) Place the culture dish in an incubator and culture it statically for about two weeks, replacing the culture medium with fresh medium every 2-3 days during this period; (4) Observe the cells. When monoclonal cell clusters visible to the naked eye are formed, stop culturing, discard the culture medium, wash with PBS, and fix the cells with 3 mL of 4% paraformaldehyde fixative for 30 min. (5) Remove the fixative, wash with PBS, and stain the cells with 0.1% crystal violet solution for 30 min; (6) Recover the crystal violet solution, rinse the culture dish slowly with running water to wash away the excess dye solution, turn the treated culture dish upside down on absorbent paper to dry, take pictures and record, use ImageJ to count the number of blue spots in each dish and conduct comparative analysis. The results are as follows: Figure 6 , Figure 7 and Figure 8 shown.

[0039] like Figure 6 , Figure 7 and Figure 8 The effect of tetrahydromagnolia on the proliferation of A549, H1299, HT29, HCT116, SW1990 and PANC-1 was detected by plate monoclonal formation experiment. The experimental results showed that with the increase of tetrahydromagnolia drug concentration, the number of cell clones decreased, and the statistical results were significantly different, indicating that tetrahydromagnolia can inhibit the proliferation of A549, H1299, HT29, HCT116, SW1990 and PANC-1.

[0040] In summary, it has strong anti-cancer activity, antioxidant and anti-aging effects on malignant tumor cells such as lung cancer, intestinal cancer and pancreatic cancer. Tetrahydromagnolia bark extract has strong proliferation inhibition activity on malignant tumor cells, thereby effectively exerting anti-tumor effects; therefore, Magnolia bark extract can be used to prepare anti-tumor drugs. Among them, anti-lung cancer drugs containing 30~90μM tetrahydromagnolia bark extract can inhibit the proliferation of lung cancer A549 cells and lung cancer H1299 cells; anti-intestinal cancer drugs containing 25~75μM tetrahydromagnolia bark extract can inhibit the proliferation of intestinal cancer HT29 cells and intestinal cancer HCT116 cells; anti-pancreatic cancer drugs containing 40~80μM tetrahydromagnolia bark extract can inhibit the proliferation of pancreatic cancer SW1990 cells and PANC-1 cells.

[0041] Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0042] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. Use of Magnolia officinalis extract as a medicinal active ingredient for preparing anti-tumor drugs.

2. The use according to claim 1, characterized in that: The magnolia bark extract is tetrahydromagnolia bark ol.

3. The use according to claim 1, characterized in that: The anti-tumor drug is selected from one or more of anti-lung cancer drugs, anti-intestinal cancer drugs and anti-pancreatic cancer drugs.

4. An anti-lung cancer drug, characterized in that: The anti-lung cancer drug contains 30-90 μM tetrahydromagnolia nobile.

5. The anti-lung cancer drug according to claim 4, characterized in that: The anti-lung cancer drug is used for inhibiting the proliferation ability of lung cancer A549 cells and lung cancer H1299 cells.

6. An anti-intestinal cancer drug, characterized in that: The anti-colon cancer drug contains 25-75 μM tetrahydromagnolia nobile.

7. The anti-intestinal cancer drug according to claim 6, characterized in that: The anti-intestinal cancer drug is used to inhibit the proliferation ability of intestinal cancer HT29 cells and intestinal cancer HCT116 cells.

8. An anti-pancreatic cancer drug, characterized in that: The anti-pancreatic cancer drug contains 40-80 μM tetrahydromagnolia nobile.

9. The anti-pancreatic cancer drug according to claim 8, characterized in that: The anti-pancreatic cancer drug is used for inhibiting the proliferation ability of pancreatic cancer SW1990 cells and PANC-1 cells.

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